Methods for targeting BCL11a enhancer functional regions for fetal hemoglobin reinduction

Abstract

To provide nucleic acid molecules targeting a BCL11A enhancer functional region, compositions comprising the nucleic acid molecules, and methods for increasing a fetal hemoglobin level in cells by disrupting BCL11A expression at the genomic level.SOLUTION: Disclosed is a nucleic acid molecule comprising a nucleic acid sequence being: a. complementary to the plus or minus strand of the human chromosome 2 at location 60725424 to 60725688 (+55 functional region); b. complementary to the plus or minus strand of the human second chromosome at location 60722238 to 60722466 (+58 functional region); or c. complementary to the plus or minus strand of the human chromosome 2 at location 60718042 to 60718186 (+62 functional region), where the human chromosome 2 is that according to UCSC Genome Browser hg 19 human genome assembly.SELECTED DRAWING: Figure 3G

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62 / 158,882, filed May 8, 2015, the contents of which are incorporated herein by reference in their entirety.

[0002] government support This invention was made with government support under Grant Nos. K08DK093705, P01HL032262, and P01HL32259 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on May 5, 2016, is named 701039-084941-PCT_SL.txt and is 73,650 bytes in size. [Background technology]

[0004] background Normal adult hemoglobin contains four globin proteins, two of which are alpha (α) proteins and two of which are beta (β) proteins. During fetal development in mammals, particularly humans, the fetus produces fetal hemoglobin, which contains two gamma (γ) globin proteins instead of two beta globin proteins. During the neonatal period, a globin switch, also known as the "fetal switch," occurs, during which erythroid progenitor cells switch from producing primarily gamma globin to producing primarily beta globin. The developmental switch from producing primarily fetal hemoglobin, or HbF (α2γ2), to producing adult hemoglobin, or HbA (α2β2), begins approximately 28 to 34 weeks of gestation and continues until shortly after birth, when HbA becomes predominant. This switch is primarily due to a decrease in transcription of the gamma globin gene and an increase in transcription of the beta globin gene.Normal adult blood contains, on average, less than 1% HbF, but residual HbF levels in healthy adults vary by more than 20-fold and are genetically controlled.

[0005] Hemoglobinopathies include several inherited anemias characterized by reduced red blood cell (RBC) production and / or increased red blood cell (RBC) breakdown (hemolysis). They also include genetic defects that result in the production of abnormal hemoglobins and a concomitant impairment of the body's ability to maintain oxygen levels. These disorders include the inability to produce sufficient amounts of normal beta-globin or the complete absence of normal beta-globin. These beta-globin protein-related disorders are commonly referred to as beta-hemoglobinopathies. For example, beta-thalassemia results from partial or complete impairment of beta-globin gene expression, resulting in abnormal or absent hemoglobin A. Sickle cell anemia results from a point mutation in the beta-globin structural gene, resulting in the production of abnormal (sickle) hemoglobin (HbS). HbS is prone to polymerization, especially under deoxygenated conditions. HbS RBCs are more fragile than normal RBCs and are more susceptible to hemolysis, ultimately leading to anemia.

[0006] In recent years, pharmacological manipulation of fetal hemoglobin (α2γ2, HbF) has attracted attention in research aimed at reducing globin chain imbalance and hemoglobin polymerization in patients with beta-hemoglobinopathies. The therapeutic potential of this approach is demonstrated by phenotypic observations in patients with both homozygous beta-thalassemia and hereditary hyperfetal hemoglobinopathy (HPFH). Furthermore, the need for transfusions is reduced in patients with homozygous beta-thalassemia who lack adult hemoglobin synthesis, when fetal hemoglobin levels are high. Furthermore, some adult patients with beta-chain abnormalities have been shown to have higher-than-normal fetal hemoglobin (HbF) levels and a milder clinical course than patients with normal adult HbF levels. For example, a group of Saudi Arabian sickle cell disease patients with 20-30% HbF expression exhibited milder clinical symptoms. It is now known that hemoglobin disorders such as sickle cell anemia and β-thalassemia can be improved by increasing HbF production.

[0007] The switch from fetal hemoglobin to adult hemoglobin (α2γ2, HbA) usually occurs within 6 months after birth. However, in the majority of patients with beta hemoglobinopathies, the upstream gamma globin gene is intact and fully functional. Therefore, reactivation of this gene may maintain functional hemoglobin synthesis in adulthood and reduce the severity of the disease. Unfortunately, the molecular mechanisms underlying this globin switch remain poorly understood.

[0008] Evidence that reactivating fetal hemoglobin production is feasible comes from experiments demonstrating the generation of erythroid colonies and erythroid bursts in semisolid medium when peripheral blood containing clonogenic cells is treated with appropriate growth factor combinations. Individual cells in such colonies can accumulate fetal hemoglobin (HbF), adult hemoglobin (HbA), or both. In adult blood cultures, nucleated red blood cells can accumulate either HbA alone (F-A+) or a combination of HbF and HbA (F+A+). Importantly, each colony contains both F+ and F- cells, indicating that both cell types are descendants of the same circulating stem cell. Thus, during early development in culture, cells are selectively conditioned to express or not express HbF by a currently unknown mechanism. The proportion of adult F+ cells generated in culture appears to be dependent on culture conditions rather than being preprogrammed in vivo. A shift to a pathway that expresses both HbF and HbA can be achieved in vitro, for example, by increasing serum concentrations, which is due to the action of an unidentified substance that adsorbs to activated charcoal.

[0009] A distal regulatory region upstream of the BCL11A gene was recently discovered that can regulate the expression of the BCL11A protein. The BCL11A protein acts as a stage-specific regulator of fetal hemoglobin expression by suppressing γ-globin induction. This upstream distal regulatory region was mapped to positions 60,716,189–60,728,612 on human chromosome 2 in the UCSC Genome Browser hg19 human genome assembly. Notably, this upstream distal regulatory region consistently contains three DNAse 1-hypersensitive sites (DHS) at +62, +58, and +55. Identification of specific functional regions within this approximately 12 kb molecule that play a role in the globin switch is important for developing novel therapeutic strategies to interfere with adult hemoglobin and induce fetal hemoglobin synthesis. Such functional regions would provide new targets for the development of therapeutic interventions for various hemoglobinopathies, where reactivation of fetal hemoglobin synthesis would significantly improve disease severity and morbidity. Summary of the Invention

[0010] overview The embodiments described herein are based in part on the discovery of defined functional regions within the approximately 12 kb enhancer region of BCL11A that regulate BCL11A protein expression. These functional regions map to three previously identified DNAse 1-hypersensitive sites (DHSs) +62, +58, and +55. Specifically, functional regions are found on human chromosome 2 at positions 60725424-60725688 (+55 functional region); 60722238-60722466 (+58 functional region); and 60718042-60718186 (+62 functional region). Disruption of genome editing in these regions was functionally validated for BCL11A mRNA expression, BCL11A protein expression, and ultimately, enrichment of the resulting fetal hemoglobin (HbF). Using CRISPR / Cas9 technology, small single guide RNA (sgRNA) sequences are designed to target these functional regions, and this disruption results in a normalized HbF enrichment of at least 0.259 or more.In particular, targeting and disrupting the +58 functional region results in an extremely high HbF enrichment, while targeting and disrupting the +55 or +62 functional region results in a moderate HbF enrichment.Therefore, using specifically designed sgRNA and CRISPR technology to target these three +62, +58 and +55 functional regions, either alone or in combination, can provide a therapeutic strategy for interfering with adult hemoglobin and inducing fetal hemoglobin synthesis.

[0011] Provided herein are nucleic acid molecules that target three BCL11A enhancer functional regions (these three +62, +58 and +55), compositions that comprise said nucleic acid molecules, and the method for increasing fetal hemoglobin levels in cells by disrupting BCL11A expression at genome level.Also provided herein are methods and compositions for treating hemoglobinopathy by reinducing fetal hemoglobin levels.In particular, said nucleic acid molecules target +62, +58 and / or +55 enhancer functional regions.

[0012] Thus, in one aspect, provided herein is a nucleic acid molecule comprising a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, and the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the entire genomic DNA sequence on human chromosome 2 at positions 60,716,189-60,728,612.

[0013] In one aspect, provided herein is a nucleic acid molecule consisting essentially of a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, and wherein the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the entire genomic DNA sequence on human chromosome 2 at positions 60,716,189-60,728,612.

[0014] In one aspect, the disclosure provides a vector comprising a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, and wherein the nucleic acid sequence excludes the entire human chromosome 2 and also excludes genomic DNA sequences on human chromosome 2 at positions 60,716,189-60,728,612.

[0015] In one aspect, the disclosure provides a vector consisting essentially of a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, and wherein the nucleic acid sequence excludes human chromosome 2 in its entirety and also excludes genomic DNA sequences on human chromosome 2 at positions 60,716,189-60,728,612.

[0016] In some embodiments, the present disclosure provides a composition comprising the above-mentioned nucleic acid molecule and / or the above-mentioned vector.In one embodiment, the composition is used in an in vitro method (for example, transfection with the described nucleic acid and / or vector, or genetic modification as described herein) to produce modified cells, so that the cells have reduced or decreased BCL11A mRNA or protein expression compared to similar cells that have not undergone the modification process.

[0017] In one aspect, the disclosure provides a method of increasing fetal hemoglobin levels in a cell, the method comprising contacting an isolated cell with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the cell or its progeny compared to the cell prior to the contacting, wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly. In one embodiment, the method is an in vitro or ex vivo method.

[0018] In one embodiment, the disclosure provides an isolated genetically modified human cell having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) according to the methods described herein. In one embodiment, the isolated genetically modified human cell has reduced or decreased BCL11A mRNA or protein expression compared to a control cell that does not have any genetic modifications on chromosome 2 at positions 60,716,189-60,728,612.

[0019] In one aspect, the disclosure provides a method for producing an isolated genetically modified human cell having at least one genetic modification, the method comprising contacting the isolated cell with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, wherein the human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly.

[0020] In one aspect, the disclosure provides a method for producing progenitor cells having reduced BCL11A mRNA or protein expression, the method comprising contacting isolated progenitor cells with a nucleic acid molecule described herein or a vector described herein.

[0021] In one aspect, the disclosure provides a method for producing progenitor cells with reduced BCL11A mRNA or BCL11A protein expression, comprising contacting isolated progenitor cells with an agent that binds to a human BCL11A enhancer functional region located on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region). wherein the agent binds to (a) the plus or minus strand of human chromosome 2 at positions 60725424-60725688 (+55 functional region); (b) the plus or minus strand of human chromosome 2 at positions 60722238-60722466 (+58 functional region); or (c) the plus or minus strand of human chromosome 2 at positions 60718042-60718186 (+62 functional region); wherein human chromosome 2 is according to the UCSC Genome Browser hg 19 human genome assembly, thereby reducing BCL11A mRNA or protein expression.

[0022] In one aspect, the disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising contacting isolated hematopoietic progenitor cells in the mammal with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting, wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly.

[0023] In one aspect, the present disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, the method comprising transplanting into the mammal an isolated genetically modified human cell described herein or a composition described herein.

[0024] Another aspect described herein relates to the use of an isolated genetically modified human cell having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) according to the methods described herein for the purpose of increasing fetal hemoglobin levels in a mammal.

[0025] Another aspect described herein relates to the use of isolated genetically modified human cells having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) according to the methods described herein for the treatment of a hemoglobinopathy in a mammal.

[0026] Another aspect described herein relates to the use of an isolated genetically modified human cell having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) according to the methods described herein for the manufacture of a medicament for the treatment of a hemoglobinopathy in a mammal, whereby fetal hemoglobin levels are increased in the mammal.

[0027] Another aspect described herein is a composition comprising an isolated genetically modified human cell having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) according to the methods described herein. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier.

[0028] Another aspect described herein relates to the use of a composition comprising isolated genetically modified human cells having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) according to the methods described herein for the purpose of increasing fetal hemoglobin levels in a mammal.

[0029] Another aspect described herein relates to the use of a composition comprising isolated genetically modified human cells having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) according to the methods described herein for the treatment of a hemoglobinopathy in a mammal.

[0030] Another aspect described herein relates to the use of a composition comprising isolated genetically modified human cells having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) according to the methods described herein for the manufacture of a medicament for the treatment of a hemoglobinopathy in a mammal, thereby increasing fetal hemoglobin levels in the mammal.

[0031] Another aspect described herein is a composition comprising a nucleic acid molecule described herein or a vector described herein together with a vector carrying at least one DNA-targeting endonuclease or a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves human cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier.

[0032] Another aspect described herein relates to the use of a composition comprising a nucleic acid molecule described herein or a vector described herein together with a vector carrying at least one DNA-targeting endonuclease or a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves human cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, for the purpose of increasing fetal hemoglobin levels in a mammal.

[0033] Another aspect described herein relates to the use of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves human cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, for the treatment of a hemoglobinopathy in a mammal.

[0034] Another aspect described herein relates to the use of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves human cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, for the manufacture of a medicament for the treatment of a hemoglobinopathy in a mammal, whereby fetal hemoglobin levels in the mammal are increased.

[0035]

[0013] In one aspect, provided herein is the use of a nucleic acid molecule comprising a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is identified by the UCSC Genome Browser hg 19 human genome assembly, wherein the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the entire genomic DNA sequence on human chromosome 2 at positions 60,716,189 to 60,728,612. Use of a nucleic acid molecule that reduces BCL11A mRNA or protein expression for increasing fetal hemoglobin in a mammal, or for treating a hemoglobinopathy in a mammal, or for reducing BCL11A mRNA or expression.

[0036] In one aspect, provided herein is use of an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves human cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, for increasing fetal hemoglobin in a mammal, or for treating a hemoglobinopathy in a mammal, or for reducing BCL11A mRNA or expression.

[0037] In one embodiment, provided herein is the use of an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting enzyme or a vector carrying a coding sequence for a DNA-targeting enzyme, wherein the DNA-targeting enzyme makes at least one epigenetic modification in human cellular genomic DNA on chromosome 2, thereby affecting BCL11A mRNA or expression, for increasing fetal hemoglobin in a mammal, or for treating a hemoglobinopathy in a mammal, or for reducing BCL11A mRNA or expression. In one embodiment, the at least one epigenetic modification is at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region). In another embodiment, the effect of one epigenetic modification is to reduce BCL11A mRNA or protein expression. In one embodiment, at least one epigenetic modification in the cellular genomic DNA on chromosome 2 indirectly or directly affects positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) on chromosome 2.

[0038] In one aspect, provided herein is a use of any of the isolated cells described herein for increasing fetal hemoglobin in a mammal or for treating a hemoglobinopathy in a mammal.

[0039] In one aspect, provided herein is use of a composition comprising isolated genetically modified human cells for increasing fetal hemoglobin in a mammal or for treating a hemoglobinopathy in a mammal, wherein the cells have a sequence encoding a gene encoding a gene encoding a gene for the hemoglobin-dependent gene of chromosome 2 between positions 60725424 and 60725688 (+55 functional region), and / or between positions 60722238 and 60722466 (+58 functional region), and / or between positions 60718042 and 60718186 (+62 functional region) (UCSC Genome Browser hg and at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly) created by a process of contacting a cellular genomic DNA on chromosome 2 (according to the UCSC Genome Browser hg 19 human genome assembly) with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves the cellular genomic DNA on chromosome 2 (according to the UCSC Genome Browser hg 19 human genome assembly) causing at least one genetic modification therein.

[0040] In one aspect, provided herein is the use of a composition comprising isolated genetically modified human cells for increasing fetal hemoglobin in a mammal or for treating a hemoglobinopathy in a mammal, wherein the cells have at least one epigenetic modification on chromosome 2. In one aspect, the at least one epigenetic modification on chromosome 2 is at positions 60725424-60725688 (the +55 functional region), and / or positions 60722238-60722466 (the +58 functional region), and / or positions 60718042-60718186 (the +62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly). In another embodiment, the at least one epigenetic modification on chromosome 2 is made by a process of contacting the cell with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA targeting enzyme or a vector carrying a coding sequence for a DNA targeting enzyme, wherein the DNA targeting enzyme effects and causes at least one epigenetic modification in the cellular genomic DNA on chromosome 2 affecting positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly).

[0041] In one aspect, provided herein is a use of any isolated cell described herein or any one of the compositions described herein for the manufacture of a medicament for increasing fetal hemoglobin in a mammal in need thereof, or for the treatment of a hemoglobinopathy in a mammal.

[0042] Another aspect described herein is a method of increasing fetal hemoglobin levels in a cell, comprising contacting an isolated cell with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the cell or its progeny compared to the cell prior to the contacting.

[0043] Another aspect described herein is a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising contacting isolated hematopoietic progenitor cells in the mammal with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting.

[0044] Another aspect described herein is a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising transplanting into the mammal isolated genetically modified human cells having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region).

[0045] In one aspect, the disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising providing an isolated population of hematopoietic progenitor or stem cells from the mammal ex vivo, and contacting the population of hematopoietic progenitor or stem cells with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting.

[0046] In one aspect, the disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising isolating a population of hematopoietic progenitor or stem cells from the mammal and ex vivo transfecting cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) with a DNA targeting enzyme. contacting a population of hematopoietic progenitor or stem cells with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting.

[0047] In one aspect, the disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising the steps of: (a) providing for isolating a population of hematopoietic progenitor cells or hematopoietic stem cells from the mammal; and (b) deleting / adding / substituting cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) to cause at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting.

[0048] In one aspect, the disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising isolating a population of hematopoietic progenitor or stem cells from the mammal and deleting ex vivo cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting.

[0049] In one aspect, the disclosure provides a method of treating a hemoglobinopathy in a mammal, comprising the steps of: (a) providing hematopoietic progenitor cells or hematopoietic stem cells or iPSCs; (b) ex vivo or in vitro DNA targeting of cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region). contacting the cells with an effective amount of a composition comprising at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting; and (c) administering the cells of step (b) to the mammal.

[0050] In one aspect, the disclosure provides a method of treating a hemoglobinopathy in a mammal, the method comprising: (a) isolating hematopoietic progenitor cells or hematopoietic stem cells from the mammal; (b) contacting the cells, ex vivo or in vitro, with an effective amount of a composition comprising at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting; and (c) administering the cells of step (b) to the mammal.

[0051] In one aspect, the disclosure provides a method of treating a hemoglobinopathy in a mammal, the method comprising: (a) providing hematopoietic progenitor cells or hematopoietic stem cells or iPSCs; (b) deleting cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) ex vivo to cause at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting; and (c) administering the cells of step (b) to the mammal.

[0052] In one aspect, the disclosure provides a method of treating a hemoglobinopathy in a mammal, the method comprising: (a) isolating hematopoietic progenitor cells or hematopoietic stem cells from the mammal; (b) deleting cellular genomic DNA ex vivo on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting; and (c) administering the cells of step (b) to the mammal.

[0053] In one aspect, the disclosure provides a method of treating a hemoglobinopathy in a mammal (e.g., a human), comprising introducing a composition described herein comprising an isolated genetically modified cell having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), whereby fetal hemoglobin expression is increased in the mammal.

[0054] In one aspect, the disclosure provides a method of treating a hemoglobinopathy in a mammal (e.g., a human), comprising increasing fetal hemoglobin expression in the mammal by a method described herein.

[0055] In one aspect, the present disclosure provides a composition comprising the isolated genetically modified human cell described herein.

[0056] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence excludes the entire BCL11A enhancer functional region.

[0057] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence excludes SEQ ID NOs: 136, 137, and / or 138 identified in Table 8 in their entirety.

[0058] In one embodiment of this and all other aspects described herein, the nucleic acid sequence is short and is 13 base pairs (bp) or longer. In other embodiments, the nucleic acid sequence is short and is 15 bp or longer, 16 bp or longer, 17 bp or longer, 18 bp or longer, 19 bp or longer, 20 bp or longer, 21 bp or longer, 22 bp or longer, 23 bp or longer, 24 bp or longer, 25 bp or longer, 26 bp or longer, 27 bp or longer, or 28 bp or longer.

[0059] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence is about 13-30 bp.In other embodiments, the nucleic acid sequence is about 13-20 bp, 13-21 bp, 13-22 bp, 13-23 bp, 13-24 bp, 13-25 bp, 13-26 bp, 13-27 bp, 13-28 bp, 13-29 bp, 14-20 bp, 14-21 bp, 14-22 bp, 14-23 bp, 14-24 bp, 14-25 bp, 14-26 bp, 14-27 bp, 14-28 bp, 14-29 bp, 15-20 bp, 15-21 bp, 15-22 bp, 15-23 bp, 15-24 bp, 15-25 bp, 15-26 bp, 15-27 bp, 15-28 bp, bp, 15~29bp, 16~20bp, 16~21bp, 16~22bp, 16~23bp, 16~24bp, 16~25bp, 16~26bp, 16~27bp, 16~28bp, 16~29bp, 17~20bp, 17~21bp, 17~22bp, 17~23bp, 17~24bp, 17~25bp, 17~26bp, 17~27bp, 17~28bp, 17~29bp, 18~20bp, 18~21bp, 18~22bp, 18~23bp, 18~24bp, 18~25bp, 18~26bp, 18~27bp, 18~28bp, 18~ 29bp, 19~21bp, 19~22bp, 19~23bp, 19~24bp, 19~25bp, 19~26bp, 19~27bp, 19~28bp, 19~29bp, 20~22bp, 20~23bp, 20~24bp, 20~25bp, 20~26bp, 20~27 bp, 20~28bp, 20~29bp, 21~23bp, 21~24bp, 21~25bp, 21~26bp, 21~27bp, 21~28bp, 21~29bp, 22~24bp, 22~25bp, 22~26bp, 22~27bp, 22~28bp, 22~29bp, 23-25bp, 23-26bp, 23-27bp, 23-28bp, 23-29bp, 24-26bp, 24-27bp, 24-28bp, 24-29bp, 25-27bp, 25-28bp, 25-29bp, 26-28bp, 26-29bp, 27-29bp, 14-30bp, 15-30bp, 16-30bp, 17-30bp, 18-30bp, 19-30bp, 20-30bp, 21-30bp, 22-30bp, 23-30bp, 24-30bp, 25-30bp, 26-30bp, 27-30bp, or 28-30bp.

[0060] In one embodiment of this and all other aspects described herein, the nucleic acid sequence is about 20 bp. In other embodiments, the nucleic acid sequence is about 13 bp, about 14 bp, about 15 bp, about 16 bp, about 17 bp, about 18 bp, about 19 bp, about 20 bp, about 21 bp, about 22 bp, about 23 bp, about 24 bp, about 25 bp, about 26 bp, about 27 bp, about 28 bp, about 29 bp, or about 30 bp.

[0061] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 1-94.

[0062] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence consists essentially of a sequence selected from the group consisting of SEQ ID NOs: 1-94.

[0063] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence is a sequence selected from the group consisting of SEQ ID NOs: 1-94.

[0064] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence consists of a sequence selected from the group consisting of SEQ ID NOs: 1-94.

[0065] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence further comprises a trans-activating CRISPR RNA (tracrRNA) sequence.

[0066] In one embodiment of this aspect and all other aspects described herein, the nucleic acid molecule is a single guide RNA (sgRNA).

[0067] In one embodiment of this aspect and all other aspects described herein, the nucleic acid molecule comprises a vector.

[0068] In one embodiment of this aspect and all other aspects described herein, the vector is a viral vector, e.g., a lentiviral vector.

[0069] In one embodiment of this aspect and all other aspects described herein, the vector is an sgRNA expression vector.

[0070] In one embodiment of this aspect and all other aspects described herein, the method further comprises contacting the isolated progenitor cells with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease.

[0071] In one embodiment of this aspect and all other aspects described herein, the at least one DNA-targeting endonuclease is a Cas (CRISPR-associated) protein.

[0072] In one embodiment of this aspect and all other aspects described herein, the Cas protein is Cas9.

[0073] In one embodiment of this aspect and all other aspects described herein, the isolated progenitor cell or isolated cell is a hematopoietic progenitor cell or stem cell.

[0074] In one embodiment of this aspect and all other aspects described herein, the hematopoietic precursors are cells of the erythroid lineage.

[0075] In one embodiment of this aspect and all other aspects described herein, the isolated progenitor cell or isolated cell is an induced pluripotent stem cell.

[0076] In one embodiment of this aspect and all other aspects described herein, the isolated progenitor cell or isolated cell is contacted ex vivo or in vitro.

[0077] In one embodiment of this aspect and all other aspects described herein, the contacted progenitor cell or the contacted cell acquires at least one genetic modification.

[0078] In one embodiment of this aspect and all other aspects described herein, the at least one genetic modification is a deletion, insertion, or substitution of a nucleic acid sequence.

[0079] In one embodiment of this aspect and all other aspects described herein, the at least one genetic modification is located on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region).

[0080] In one embodiment of this aspect and all other aspects described herein, the contacted progenitor cell or the contacted cell acquires at least one epigenetic modification in a BCL11A enhancer functional region.

[0081] In one embodiment of this aspect and all other aspects described herein, the at least one epigenetic modification is selected from the group consisting of alterations in DNA methylation, histone tail modifications, histone subunit composition, and nucleosome positioning.

[0082] In one embodiment of this aspect and all other aspects described herein, the at least one epigenetic modification is located on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region).

[0083] In one embodiment of this aspect and all other aspects described herein, the isolated cell or population of isolated cells is a human cell.

[0084] In one embodiment of this aspect and all other aspects described herein, the isolated cell or population of isolated cells is a progenitor cell.

[0085] In one embodiment of this aspect and all other aspects described herein, the human cells are hematopoietic progenitor cells.

[0086] In one embodiment of this aspect and all other aspects described herein, the human cells are induced pluripotent stem cells.

[0087] In one embodiment of this aspect and all other aspects described herein, the induced pluripotent stem cells are hematopoietic progenitor cells.

[0088] In one embodiment of this aspect and all other aspects described herein, the hematopoietic precursors are cells of the erythroid lineage.

[0089] In one embodiment of this aspect and all other aspects described herein, the hematopoietic progenitor cells or isolated cells are contacted ex vivo or in vitro or in vivo.

[0090] In one embodiment of this aspect and all other aspects described herein, at least one genetic modification is a deletion.

[0091] In another embodiment of this aspect and all other aspects described herein, the nucleic acid molecule consists essentially of one or more of the sequences set forth in Table 7 or SEQ ID NOs:1-94.

[0092] In a further embodiment of any of the methods of treatment, the method comprises chemotherapy and / or radiation therapy to eliminate or reduce endogenous hematopoietic progenitor or stem cells in the mammal.

[0093] In one embodiment of either method, the contacted cells having at least one genetic modification can be cryopreserved and stored until needed for administration to a mammal.

[0094] In one embodiment of any of the methods described, hematopoietic progenitor or stem cells or isolated cells can be replaced with iPSCs described herein.

[0095] In one embodiment of any of the methods described, the hematopoietic progenitor or stem cells or iPSCs or isolated cells are autologous to the mammal, meaning that the cells are derived from the same mammal. In another embodiment of the methods described, the hematopoietic progenitor or stem cells or iPSCs or isolated cells are non-autologous to the mammal, meaning that the cells are not derived from the same mammal, but from another mammal of the same species. For example, the mammal is a human.

[0096] In one embodiment of any of the methods of treatment, the method further comprises the step of selecting a mammal in need of increased fetal hemoglobin expression.

[0097] In one embodiment of any of the methods of treatment, the method further comprises the step of selecting a mammal in need of treatment for a hemoglobinopathy.

[0098] In any embodiment of any of the methods of treatment described, the hemoglobinopathy is alpha hemoglobinopathy.

[0099] In any embodiment of any of the methods of treatment described, the hemoglobinopathy is β-thalassemia.

[0100] In any embodiment of any of the methods of treatment described, the hemoglobinopathy is sickle cell anemia. [Brief explanation of the drawings]

[0101] [Figure 1] Figure 1A shows the requirement of human erythroid enhancers for BCL11A expression and HbF repression. Figure 1A shows a schematic of the human BCL11A locus (transcribed from right to left) with erythroid chromatin marks and trait-associated haplotypes indicated. Figure 1B shows ranked enhancers in primary human adult erythroid progenitors by H3K27ac signal intensity, with super-enhancers shaded. Figures 1C-1E show that deletion of the human composite BCL11A enhancer in HUDEP-2 cells demonstrates that the enhancer is required for BCL11A expression normalized to GAPDH, repression of γ-globin mRNA, and repression of HbF. Error bars reflect the standard error of the mean (SEM). [Figure 2A] Figure 1 shows representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen. Figure 2 shows a schematic diagram of the CRISPR-Cas9 enhancer screen workflow, showing library synthesis, delivery, and analysis. [Figure 2B] Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen is shown, showing sgRNA library composition by target sequence and PAM restriction. [Figure 2C] Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen showing the distribution of NGG PAM sgRNAs mapped to genomic break locations. [Figure 2D] Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen is shown. Distances to adjacent genomic cut positions for the NGG PAM sgRNA are shown. [Figure 2E] Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen is shown. HbF sorting of library-transduced cells is shown. [Figure 2F]Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen are shown. The effect of a control sgRNA on HbF enrichment is shown. Boxes indicate the first, median, and third quartiles, and whiskers indicate minimum and maximum values. ****P<0.0001, ns not significant. [Figure 2G] Representative data from an in situ tiled pool CRISPR-Cas9 BCL11A enhancer screen are shown, including dotted lines at x=y and x=8y, showing plasmid pools and cells at the end of the experiment (left), and sgRNA presentation in high and low HbF pools (right). [Figure 2H] Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen is shown. A quantile-quantile plot of sgRNA enrichment scores is shown. [Figure 3A] Functional mapping of the human BCL11A enhancer is shown. Mapping of sgRNA enrichment scores relative to genomic cleavage locations is shown. Non-targeting sgRNAs were mock-mapped at 5 bp intervals. [Figure 3B] Figure 1 shows functional mapping of human BCL11A enhancers. Correlation between extinction and enrichment scores is shown. [Figure 3C] Functional mapping of the human BCL11A enhancer is shown. BCL11A expression normalized to GAPDH is shown in HUDEP-2 cells harboring individual DHS deletions or inversions. [Figure 3D] Functional mapping of the human BCL11A enhancer is shown. β-like globin expression is shown in HUDEP-2 cells with individual DHS deletions or inversions. [Figure 3E] Functional mapping of the human BCL11A enhancer is shown. HbF+ fractions are shown in HUDEP-2 cells with individual DHS deletions or inversions. [Figure 3F]Functional mapping of the human BCL11A enhancer is shown. Correlation between HbF enrichment scores from the pooled sgRNA screen and the HbF fraction from array validation of individual sgRNAs in HUDEP-2 cells is shown. [Figure 3G] Figures 3G-3I show functional mapping of the human BCL11A enhancer. BCL11A expression, β-like globin expression, and HbF+ fraction normalized to GAPDH are shown in HUDEP-2 cells derived from primary human erythroid progenitors transduced with Cas9 and individual sgRNAs. Error bars represent SEM. Filtering for enrichment scores of >0.259 and enrichment scores of human library-targeting sgRNAs for NGG RC and NGG sgRNA yielded 135 target sequences, shown in Table 7. These are sgRNAs targeting the +62, +58, and +55 functional regions in the BCL11A enhancer, as well as a set of sgRNAs targeting exon 2 of BCL11A. [Figure 3H] See legend to Figure 3G. [Figure 3I] See legend to Figure 3G. [Figure 4A] Figures 4A-4C show genomic features and relative inferred functional enhancer states. Hidden Markov model (HMM) segmentation of functional enhancer states. HbF enrichment scores are shown across DHS +55, +58, and +62 by gray lines and circles, and the curved graph line represents smooth enrichment scores. DNase I sequencing from primary human erythroblasts. PhyloP (scale from -4.5 to 4.88) and PhastCons (0 to 1) estimate evolutionary conservation among 100 vertebrate species. [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 5A] Functional sequence requirements in the mouse BCL11A erythroid enhancer for in vivo hemoglobin switching are shown. Mapping of sgRNA εy:mCherry enrichment scores to genomic cleavage locations is shown. Non-targeting sgRNAs were pseudo-mapped at 5 bp intervals. [Figure 5B] Figure 1 shows the functional sequence requirement in the mouse BCL11A erythroid enhancer for in vivo hemoglobin switching. Figure 2 shows BCL11A expression in mouse erythroid clones harboring individual DHS deletions or inversions, normalized to the control set at 1. [Figure 5C] Functional sequence requirements for the mouse BCL11A erythroid enhancer for in vivo hemoglobin switching are shown. HMM segmentation of the active functional state in the +62 ortholog is shown. Enrichment scores are shown as gray lines and circles; the curved graph within is a smoothed enrichment score. DNase I sequencing from mouse fetal liver erythroid precursors. BCL11A expression determined by RT-qPCR is displayed as a heatmap in 108 hemizygous +62 ortholog deletion clones, listed from top to bottom by the genomic location of the deletion midpoint. PhyloP (scale -3.3 to 2.1) and PhastCons (0 to 1) estimate evolutionary conservation among 30 vertebrate species. [Figure 5D] Figure 1 shows the functional sequence requirement in the mouse BCL11A erythroid enhancer for in vivo hemoglobin switching. Figure 2 shows transgenic human globin expression in mouse E16.5 chimeric β-YAC / +62 deleted fetal liver. [Figure 5E] Figures 5E-5F show the functional sequence requirement in the mouse BCL11A erythroid enhancer for in vivo hemoglobin switching. BCL11A expression, B cell numbers, and transgenic human β-like globin expression in β-YAC / +62 deletion mice are shown. *P<0.05. Error bars represent SEM. [Figure 5F] See legend to Figure 5E. [Figure 6A]Figures 6A-6F summarize representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen. Distribution of NAG PAM sgRNAs mapped to genomic cleavage locations. Vertical lines represent sgRNA cleavage sites for sgRNAs mapped to the plus and minus strands. Distance to adjacent genomic cleavage locations for NAG PAM sgRNAs. Deep sequencing of the lentiviral plasmid library demonstrates that 1,337 of 1,338 sgRNAs (99.9%) were successfully cloned. The representation of sgRNAs within the library shows a relatively narrow distribution, with a median of 718, and the 10th and 90th percentiles ranging from 337 to 1,205 normalized reads, as indicated by the vertical dotted lines. HbF distribution in HUDEP-2 cells transduced with Cas9 and individual sgRNAs (either non-targeting or targeting BCL11A exon 2). Enrichment scores for NGG sgRNAs across six biological replicates. Mapping of sgRNA loss scores for NGG sgRNAs relative to genomic cleavage locations and repetitive elements. Non-targeting sgRNAs were mock-mapped at 5 bp intervals. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 6F] See legend to Figure 6A. [Figure 7A] Figure 1 shows validation of select sgRNAs identified in the described enhancer screen. The HbF+ fraction in HUDEP-2 cells transduced in array format with 24 sgRNAs from all five mapping categories whose enrichment scores ranged from highest to lowest in the screen. [Figure 7B]Validation of selected sgRNAs identified in the described enhancer screen is shown. β-like globin gene expression normalized to a reference gene (GAPDH) is shown in primary human erythroid precursors transduced with Cas9 and individual sgRNAs. Erythroid differentiation of primary human erythroid precursors was assessed by CD71 and CD235a surface markers, enucleation frequency (CD235a+Hoescht33342-), and morphology by May-Grünwald-Giemsa staining. [Figure 8A] Functionality assessment of enhancer sequences. Hidden Markov model (HMM) topology based on Gaussian emission of sgRNA enrichment scores is shown, which is used to infer three functional enhancer states (active, repressed, and neutral). All possible transitions between states are allowed. [Figure 8B] Functional evaluation of enhancer sequences is shown. The frequency distribution of indels from HUDEP-2 cells exposed to Cas9 and individual sgRNAs, sorted into high and low HbF pools, and subjected to deep sequencing of the target sites is shown. Indels were calculated per nucleotide across the amplicon surrounding the sgRNA-1617 and sgRNA-1621 cleavage sites (dotted lines). The indel enrichment ratio was calculated by dividing the normalized indel frequency in the high HbF pool by the low HbF pool. [Figure 9A] Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen is shown. A schematic diagram of the mouse BCL11A locus (transcribed left to right) is shown, with erythroid chromatin marks and regions of primary sequence homology to human DHS indicated. [Figure 9B] Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen are shown, ranking enhancers in mouse fetal liver erythroid precursors by H3K27ac signal intensity, with super-enhancers shaded. [Figure 9C]Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen showing mCherry expression in MEL εy:mCherry reporter cells upon exposure to Cas9 and individual sgRNAs targeting Bcl11a exon 2. [Figure 9D] Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen is shown. A representative strategy for knocking in the fluorescent protein mCherry into the mouse embryonic globin Hbb-y locus (encoding the εy embryonic globin chain) by homologous recombination is shown. [Figure 9E] Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen is shown, showing sgRNA library composition by target sequence and PAM restriction. [Figure 9F] Figures 9F-9G show representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen. The distribution of NGG (top left) and NAG (top right) PAM sgRNAs mapped to genomic cleavage locations is shown. Vertical lines represent sgRNA cleavage sites for sgRNAs mapped to the plus and minus strands. Distance to adjacent genomic cleavage locations for NGG (bottom left) and NAG (bottom right) PAM sgRNAs. [Figure 9G] See legend to Figure 9F. [Figure 9H] Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen is shown. [Figure 9I]Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen are shown. Deep sequencing of the lentiviral plasmid library demonstrated that 1,271 of 1,271 (100%) sgRNAs were successfully cloned. The representation of sgRNAs within the library showed a relatively narrow distribution, with a median of 735, and the 10th and 90th percentiles ranging from 393 to 1,240 normalized reads, as indicated by the vertical dotted lines. [Figure 9J] Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen is shown, showing εy:mCherry sorting of library-transduced cells. [Figure 9K] Representative data from an in situ tiled pooled CRISPR-Cas9 BCL11A enhancer screen are shown. Control sgRNA enrichment is shown. Boxes indicate the first, median, and third quartiles, and whiskers indicate minimum and maximum values. ****P<0.0001. [Figure 10A] Figure 1 shows a BCL11A enhancer screen analysis, with dotted lines at x=y and x=8y indicating plasmid pools and cells at the end of the experiment (left) and sgRNA presentation in high εy:mCherry and low εy:mCherry pools (right). [Figure 10B] Figure 1 shows a BCL11A enhancer screen analysis. Quantile-quantile plots of sgRNA enrichment scores are shown. [Figure 10C] Figure 1 shows a BCL11A enhancer screen analysis. Mapping of sgRNA loss scores for NGG sgRNAs relative to genomic cut locations and repetitive elements is shown. Non-targeting sgRNAs were mock-mapped at 5 bp intervals. [Figure 10D] Figure 1 shows a BCL11A enhancer screen analysis, showing the correlation between loss and enrichment scores. [Figure 11]Figure 11A shows the requirement for the BCL11A erythroid enhancer during murine ontogeny. Figure 11A shows BCL11A expression determined by RT-qPCR in 108 hemizygous +62 ortholog deletion clones. The average effective size per nucleotide unit was calculated as the average fold change in BCL11A expression across all clones in which that nucleotide was deleted. Gray shading represents one standard deviation. Figure 11B shows an analysis scheme of transgenic human β-like globin (β-YAC) gene expression during chimeric fetal liver development. The right panel shows data from a control β-YAC chimeric fetal liver demonstrating that γ-globin suppression occurs by E16.5. Figure 11C shows the progeny of a heterozygous BCL11A +62 ortholog deletion intercross compared to the expected Mendelian ratio. Figure 11D shows BCL11A expression relative to GAPDH in E16.5 mouse brains from various genotypes. Fetal liver fractions consisting of B cell precursors at E16.5 from various genotypes. Analysis of peripheral blood from 4-week-old mice examining the frequency of various circulating hematopoietic lineages in wild-type, heterozygous, and homozygous mice lacking the BCL11A+62 orthologue. [Figure 12] HUDEP-2 cells expressing dCas9-KRAB+ indicated sgRNAs were analyzed for gene expression. BCL11A is plotted relative to GAPDH. HBG is plotted relative to total beta-like globin. Two different sgRNAs (BCL_01617 and BCL_01621) targeting BCL11A+58 lead to transcriptional repression of BCL11A and derepression of HBG (gamma-globin). NT, non-targeting control. DETAILED DESCRIPTION OF THE INVENTION

[0102] Simple enumeration of tables Table 1: sgRNA sequences (Table 2) Oligonucleotide primers for deletion clone screening. (Table 3) Oligonucleotide primers for inverted clone screening. Table 4: Oligonucleotide primers for mouse +62 deletion analysis. Table 5: RT qPCR oligonucleotides. (Table 6) Location of BCL11A enhancer regions to target to achieve BCL11A knockdown. (Table 7) sgRNA sequences that resulted in HFb enrichment above 0259. (Table 8) Sequences of the BCL11A enhancer +62, +58, and +55 functional regions. (Table 9) NGA-restricted sgRNA sequences that resulted in HbF enrichment greater than 0.259.

[0103] Detailed Description The methods and compositions described herein relate, in part, to the discovery of more distinct functional regions within the approximately 12 kb enhancer region of BCL11A that regulate expression of the BCL11A protein. The functional regions are positions 60725424-60725688 (+55 functional region), 60722238-60722466 (+58 functional region), and 60718042-60718186 (+62 functional region) on human chromosome 2 according to the UCSC Genome Browser hg19 human genome assembly. The BCL11A protein acts as a stage-specific regulator of fetal hemoglobin expression by suppressing γ-globin induction.

[0104] Disruption of genome editing in these regions was functionally validated by measuring BCL11A mRNA expression, BCL11A protein expression, and ultimately the enrichment of fetal hemoglobin (HbF). Using CRISPR / Cas9 technology, small single-guide RNA (sgRNA) sequences targeting these functional regions were designed to reduce BCL11A expression and increase HbF expression. sgRNA sequences demonstrating disruption, with normalized HbF enrichment of at least 0.259, are shown in Table 7 and identified as SEQ ID NOs: 1-94.

[0105] In particular, targeting and disrupting the +58 functional region resulted in very high HbF enrichment, while targeting and disrupting the +55 or +62 functional region resulted in moderate HbF enrichment. Therefore, targeting these three +62, +58, and +55 functional regions, either alone or in combination, using specifically designed sgRNAs and CRISPR technology can provide a therapeutic strategy to interfere with adult hemoglobin and induce fetal hemoglobin synthesis.

[0106] Provided herein are nucleic acid molecules that target three BCL11A enhancer functional regions (these three +62, +58 and +55), compositions that comprise said nucleic acid molecules, and methods for increasing fetal hemoglobin levels in cells by disrupting BCL11A expression at genome level.Also provided herein are methods and compositions for treating hemoglobinopathy by reinducing fetal hemoglobin levels.In particular, said nucleic acid molecules target +62, +58 and / or +55 enhancer functional regions.

[0107] Therefore, the methods and compositions provided herein are novel methods for regulating γ-globin expression in red blood cells.More specifically, these activities can be utilized in the method for treating β-hemoglobinopathy by inducing γ-globin through the inhibition of BCL11A gene product.

[0108] The disclosure described herein, in one aspect, does not relate to the use of human embryos for industrial or commercial purposes or processes for cloning humans, for processes for modifying the genetic identity of the germline of humans, for modifying the genetic identity of animals that are likely to cause suffering to the animals without any substantial medical benefit to the humans or animals, nor to animals resulting from such processes.

[0109] Thus, in one aspect, provided herein is a nucleic acid molecule comprising a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, and the nucleic acid sequence excludes all of human chromosome 2 and also excludes the entire genomic DNA sequence on human chromosome 2 at positions 60,716,189-60,728,612.

[0110] Additionally, small single guide RNA (sgRNA) sequences designed to target BCL11A coding exon 2 using CRISPR / Cas9 technology also demonstrated effective disruption of BCL11A expression. sgRNA sequences demonstrating disruption, with normalized HbF enrichment of at least 0.259, are shown in Table 7 and identified as SEQ ID NOs: 95-135.

[0111] In one embodiment, provided herein is a nucleic acid molecule comprising a nucleic acid sequence that is complementary to the plus or minus strand of human BCL11A exon 2, wherein the nucleic acid sequence excludes the entire sequence of human BCL11A exon 2. In one embodiment, the nucleic acid sequence comprises SEQ ID NOs:94-135.

[0112] In one embodiment, provided herein is a nucleic acid molecule consisting essentially of a nucleic acid sequence that is complementary to the plus or minus strand of human BCL11A exon 2, wherein the nucleic acid sequence excludes the entire sequence of human BCL11A exon 2. In one embodiment, the nucleic acid sequence consists essentially of SEQ ID NOs:94-135.

[0113] In one embodiment, provided herein is a nucleic acid molecule consisting of a nucleic acid sequence that is complementary to the plus or minus strand of human BCL11A exon 2, where the nucleic acid sequence excludes the entire sequence of human BCL11A exon 2. In one embodiment, the nucleic acid sequence consists of SEQ ID NOs:94-135.

[0114] In one aspect, provided herein is a nucleic acid molecule consisting essentially of a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, and wherein the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the entire genomic DNA sequence on human chromosome 2 at positions 60,716,189-60,728,612.

[0115] In one aspect, the disclosure provides a vector comprising a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, and wherein the nucleic acid sequence excludes human chromosome 2 in its entirety and also excludes genomic DNA sequences on human chromosome 2 at positions 60,716,189-60,728,612.

[0116] In one aspect, the disclosure provides a vector consisting essentially of a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, and wherein the nucleic acid sequence excludes human chromosome 2 in its entirety and also excludes genomic DNA sequences on human chromosome 2 at positions 60,716,189-60,728,612.

[0117] In one aspect, the disclosure provides a vector comprising a nucleic acid sequence that is complementary to the plus or minus strand of human BCL11A exon 2, wherein the nucleic acid sequence excludes the entire human BCL11A exon 2 sequence.

[0118] One aspect described herein relates to a method for producing an isolated genetically modified human cell having at least one genetic modification, comprising contacting the cell with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector comprising a nucleic acid molecule described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), 60722238-60722466 (+58 functional region), and / or 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly) resulting in at least one genetic modification therein.

[0119] Another aspect provided herein relates to a method for increasing fetal hemoglobin levels in an isolated cell, the method comprising reducing BCL11A mRNA or protein expression in the cell. In one aspect, the reduction in BCL11A mRNA or protein expression is achieved by causing at least one genetic modification in the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly). In another aspect, reduction of BCL11A mRNA or protein expression is achieved by causing at least one genetic modification in cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), resulting in an epigenetic modification of gene function on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region). In this aspect, BCL11A enhancer activity located within positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) on this chromosome 2 is reduced.

[0120] A decrease in this aspect means that the enhancer activity in enhancing BCL11A mRNA or protein expression in cells is at least 5% less, at least 10% less, at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 1-fold less, at least 2-fold less, at least 5-fold less, at least 10-fold less, at least 100-fold less, at least 1000-fold less, or more, compared to control cells that have not been treated with any of the methods disclosed herein. By reduced BCL11A mRNA or protein expression in a cell is meant that protein expression is at least 5% lower, at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 1-fold lower, at least 2-fold lower, at least 5-fold lower, at least 10-fold lower, at least 100-fold lower, at least 1000-fold lower, or more, compared to control cells that have not been treated with any of the methods disclosed herein.

[0121] Another aspect provided herein relates to a method of increasing fetal hemoglobin levels in isolated cells, the method comprising providing an isolated human cell or progenitor cell, and reducing BCL11A mRNA or protein expression in the cell.

[0122] Another aspect provided herein relates to an ex vivo or in vitro method for increasing fetal hemoglobin levels in isolated cells, the method comprising providing an isolated human cell or progenitor cell and reducing BCL11A mRNA or protein expression in the cell.

[0123] Another aspect provided herein is an ex vivo or in vitro method for producing progenitor cells having reduced BCL11A mRNA or protein expression, comprising the step of contacting isolated progenitor cells with a nucleic acid molecule comprising, or consisting essentially of, a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is identified as a BCL11A mRNA or protein expression region in the UCSC Genome Browser hg 19 human genome assembly, wherein the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the entire genomic DNA sequence at positions 60,716,189 to 60,728,612 on human chromosome 2, thereby reducing BCL11A mRNA or protein expression.

[0124] Another aspect provided herein is an ex vivo or in vitro method for producing an isolated genetically modified human cell having at least one genetic modification, comprising contacting the isolated cell with an effective amount of a nucleic acid molecule comprising, or consisting essentially of, a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is identified as a chromosome 2 gene by the UCSC Genome Browser hg 19 human genome assembly, wherein the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the entire genomic DNA sequence on human chromosome 2 at positions 60,716,189 to 60,728,612, and causes at least one genetic modification therein.

[0125] Another aspect provided herein is an ex vivo or in vitro method for producing progenitor cells having reduced BCL11A mRNA or protein expression, comprising contacting isolated progenitor cells with a vector containing a nucleic acid molecule that comprises, or consists essentially of, a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is identified as a BCL11A mRNA or protein expression vector as described in the UCSC Genome Browser hg 19 human genome assembly, wherein the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the entire genomic DNA sequence at positions 60,716,189 to 60,728,612 on human chromosome 2, thereby reducing BCL11A mRNA or protein expression.

[0126] Another aspect provided herein is an ex vivo or in vitro method for producing an isolated genetically modified human cell having at least one genetic modification, comprising contacting the isolated cell with an effective amount of a vector comprising a nucleic acid molecule that comprises, or consists essentially of, a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is identified as a chromosome 2 gene by the UCSC Genome Browser hg 19 human genome assembly, wherein the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the entire genomic DNA sequence on human chromosome 2 at positions 60,716,189 to 60,728,612, and causes at least one genetic modification therein.

[0127] Another aspect provided herein relates to an ex vivo or in vitro method for producing an isolated genetically modified human cell having at least one genetic modification, the method comprising contacting the isolated cell with an effective amount of a composition comprising a nucleic acid described herein.

[0128] Another aspect provided herein relates to an ex vivo or in vitro method for producing an isolated genetically modified human cell having at least one genetic modification, the method comprising contacting the isolated cell with an effective amount of a composition comprising a vector described herein.

[0129] In some embodiments of any of the ex vivo or in vitro methods described herein, the isolated progenitor cells or isolated cells are hematopoietic progenitor cells.

[0130] In some embodiments of any of the ex vivo or in vitro methods described herein, the hematopoietic precursors are cells of the erythroid lineage.

[0131] In some embodiments of any of the ex vivo or in vitro methods described herein, the isolated progenitor cells or isolated cells are induced pluripotent stem cells.

[0132] Another aspect described herein relates to a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising reducing BCL11A mRNA or protein expression in hematopoietic progenitor cells in the mammal. In one aspect, the reduction in BCL11A mRNA or protein expression is achieved by causing at least one genetic modification in cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly). In another aspect, the reduction in BCL11A mRNA or protein expression is achieved by causing at least one epigenetic modification in cellular genomic DNA on chromosome 2. In another aspect, reduction of BCL11A mRNA or protein expression is achieved by inducing at least one epigenetic modification in cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region).

[0133] Another aspect provided herein relates to a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising transplanting genetically modified human cells as described herein into the mammal.

[0134] In one aspect, provided herein is a nucleic acid molecule comprising a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, and wherein the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the entire genomic DNA sequence on human chromosome 2 at positions 60,716,189-60,728,612.

[0135] In one aspect, provided herein is a nucleic acid molecule consisting essentially of a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, and wherein the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the entire genomic DNA sequence on human chromosome 2 at positions 60,716,189-60,728,612.

[0136] In one aspect, the disclosure provides a vector comprising a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is according to the UCSC Genome Browser hg 19 human genome assembly, and wherein the nucleic acid sequence excludes human chromosome 2 in its entirety and also excludes genomic DNA sequences on human chromosome 2 at positions 60,716,189-60,728,612.

[0137] In one aspect, the disclosure provides a vector consisting essentially of a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, and wherein the nucleic acid sequence excludes human chromosome 2 in its entirety and also excludes genomic DNA sequences on human chromosome 2 at positions 60,716,189-60,728,612.

[0138] In one aspect, the disclosure provides a method of increasing fetal hemoglobin levels in a cell, the method comprising contacting an isolated cell with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the cell or its progeny compared to the cell prior to the contacting, wherein human chromosome 2 is according to the UCSC Genome Browser hg 19 human genome assembly.

[0139] In one embodiment, the disclosure provides an isolated genetically modified human cell having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) according to the methods described herein. In one embodiment, the isolated genetically modified human cell has reduced or decreased BCL11A mRNA or protein expression compared to a control cell that does not have any genetic modifications on chromosome 2 at positions 60,716,189-60,728,612.

[0140] In one aspect, the disclosure provides a method for producing an isolated genetically modified human cell having at least one genetic modification, the method comprising contacting the isolated cell with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, wherein the human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly.

[0141] In one aspect, the disclosure provides a method for producing progenitor cells having reduced BCL11A mRNA or protein expression, the method comprising contacting isolated progenitor cells with a nucleic acid molecule described herein or a vector described herein.

[0142] In one aspect, the disclosure provides a method for producing progenitor cells with reduced BCL11A mRNA or BCL11A protein expression, comprising contacting isolated progenitor cells with an agent that binds to a human BCL11A enhancer functional region located on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region). wherein the agent binds to (a) the plus or minus strand of human chromosome 2 at positions 60725424-60725688 (+55 functional region); (b) the plus or minus strand of human chromosome 2 at positions 60722238-60722466 (+58 functional region); or (c) the plus or minus strand of human chromosome 2 at positions 60718042-60718186 (+62 functional region); wherein human chromosome 2 is according to the UCSC Genome Browser hg 19 human genome assembly, thereby reducing BCL11A mRNA or protein expression.

[0143] In one aspect, the disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising contacting isolated hematopoietic progenitor cells in the mammal with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting, wherein human chromosome 2 is according to the UCSC Genome Browser hg 19 human genome assembly.

[0144] In one aspect, the present disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, the method comprising transplanting into the mammal an isolated genetically modified human cell described herein or a composition described herein.

[0145] Another aspect described herein is a composition comprising an isolated genetically modified human cell having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) according to the methods described herein. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier.

[0146] Another aspect described herein is a composition comprising a nucleic acid molecule described herein or a vector described herein together with a vector carrying at least one DNA-targeting endonuclease or a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves human cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier.

[0147] Another aspect described herein is a method of increasing fetal hemoglobin levels in a cell, comprising contacting an isolated cell with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the cell or its progeny compared to the cell prior to the contacting.

[0148] Another aspect described herein is a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising contacting isolated hematopoietic progenitor cells in the mammal with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting.

[0149] Another aspect described herein is a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising transplanting into the mammal isolated genetically modified human cells having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region).

[0150] In one aspect, the disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising providing an isolated population of hematopoietic progenitor or stem cells from the mammal ex vivo, and contacting the population of hematopoietic progenitor or stem cells with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting.

[0151] In one aspect, the disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising isolating a population of hematopoietic progenitor or stem cells from the mammal, and contacting the population of hematopoietic progenitor or stem cells ex vivo with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting.

[0152] In one aspect, the disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising the steps of: (a) providing for isolating a population of hematopoietic progenitor cells or hematopoietic stem cells from the mammal; and (b) deleting / adding / substituting cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) to cause at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting.

[0153] In one aspect, the disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising isolating a population of hematopoietic progenitor or stem cells from the mammal and deleting ex vivo cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting.

[0154] In one aspect, the disclosure provides a method of treating a hemoglobinopathy in a mammal, comprising the steps of: (a) providing hematopoietic progenitor cells or hematopoietic stem cells or iPSCs; (b) ex vivo or in vitro DNA targeting of cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region). contacting the cells with an effective amount of a composition comprising at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting; and (c) administering the cells of step (b) to the mammal.

[0155] In one aspect, the disclosure provides a method of treating a hemoglobinopathy in a mammal, comprising: (a) isolating hematopoietic progenitor cells or hematopoietic stem cells from the mammal; (b) contacting, ex vivo or in vitro, the cells with an effective amount of a composition comprising at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting; and (c) administering the cells of step (b) to a mammal.

[0156] In one aspect, the disclosure provides a method of treating a hemoglobinopathy in a mammal, the method comprising: (a) providing hematopoietic progenitor cells or hematopoietic stem cells or iPSCs; (b) deleting cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) ex vivo to cause at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting; and (c) administering the cells of step (b) to the mammal.

[0157] In one aspect, the disclosure provides a method of treating a hemoglobinopathy in a mammal, the method comprising: (a) isolating hematopoietic progenitor cells or hematopoietic stem cells from the mammal; (b) deleting cellular genomic DNA ex vivo on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting; and (c) administering the cells of step (b) to the mammal.

[0158] In one aspect, the disclosure provides a method of treating a hemoglobinopathy in a mammal (e.g., a human), comprising introducing a composition described herein comprising an isolated genetically modified cell having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), 60722238-60722466 (+58 functional region), and / or 60718042-60718186 (+62 functional region), whereby fetal hemoglobin expression is increased in the mammal.

[0159] In one aspect, the disclosure provides a method of treating a hemoglobinopathy in a mammal (e.g., a human), comprising increasing fetal hemoglobin expression in the mammal by a method described herein.

[0160] In one aspect, the present disclosure provides a composition comprising the isolated genetically modified human cell described herein.

[0161] In one embodiment of this aspect and all other aspects described herein, the method further comprises selecting a mammal in need of increasing fetal hemoglobin levels therein.

[0162] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence excludes the entire BCL11A enhancer functional region.

[0163] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence excludes SEQ ID NOs: 136, 137, and / or 138 identified in Table 8 in their entirety.

[0164] In one embodiment of this and all other aspects described herein, the nucleic acid sequence is short and is 13 base pairs (bp) or longer. In other embodiments, the nucleic acid sequence is short and is 15 bp or longer, 16 bp or longer, 17 bp or longer, 18 bp or longer, 19 bp or longer, 20 bp or longer, 21 bp or longer, 22 bp or longer, 23 bp or longer, 24 bp or longer, 25 bp or longer, 26 bp or longer, 27 bp or longer, or 28 bp or longer.

[0165] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence is about 13-30 bp.In other embodiments, the nucleic acid sequence is about 13-20 bp, 13-21 bp, 13-22 bp, 13-23 bp, 13-24 bp, 13-25 bp, 13-26 bp, 13-27 bp, 13-28 bp, 13-29 bp, 14-20 bp, 14-21 bp, 14-22 bp, 14-23 bp, 14-24 bp, 14-25 bp, 14-26 bp, 14-27 bp, 14-28 bp, 14-29 bp, 15-20 bp, 15-21 bp, 15-22 bp, 15-23 bp, 15-24 bp, 15-25 bp, 15-26 bp, 15-27 bp, 15-28 bp, bp, 15~29bp, 16~20bp, 16~21bp, 16~22bp, 16~23bp, 16~24bp, 16~25bp, 16~26bp, 16~27bp, 16~28bp, 16~29bp, 17~20bp, 17~21bp, 17~22bp, 17~23bp, 17~24bp, 17~25bp, 17~26bp, 17~27bp, 17~28bp, 17~29bp, 18~20bp, 18~21bp, 18~22bp, 18~23bp, 18~24bp, 18~25bp, 18~26bp, 18~27bp, 18~28bp, 18~ 29bp, 19~21bp, 19~22bp, 19~23bp, 19~24bp, 19~25bp, 19~26bp, 19~27bp, 19~28bp, 19~29bp, 20~22bp, 20~23bp, 20~24bp, 20~25bp, 20~26bp, 20~27 bp, 20~28bp, 20~29bp, 21~23bp, 21~24bp, 21~25bp, 21~26bp, 21~27bp, 21~28bp, 21~29bp, 22~24bp, 22~25bp, 22~26bp, 22~27bp, 22~28bp, 22~29bp, 23-25bp, 23-26bp, 23-27bp, 23-28bp, 23-29bp, 24-26bp, 24-27bp, 24-28bp, 24-29bp, 25-27bp, 25-28bp, 25-29bp, 26-28bp, 26-29bp, 27-29bp, 14-30bp, 15-30bp, 16-30bp, 17-30bp, 18-30bp, 19-30bp, 20-30bp, 21-30bp, 22-30bp, 23-30bp, 24-30bp, 25-30bp, 26-30bp, 27-30bp, or 28-30bp.

[0166] In one embodiment of this and all other aspects described herein, the nucleic acid sequence is about 20 bp. In other embodiments, the nucleic acid sequence is about 13 bp, about 14 bp, about 15 bp, about 16 bp, about 17 bp, about 18 bp, about 19 bp, about 20 bp, about 21 bp, about 22 bp, about 23 bp, about 24 bp, about 25 bp, about 26 bp, about 27 bp, about 28 bp, about 29 bp, or about 30 bp.

[0167] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence comprises a sequence selected from the group consisting of SEQ ID NOs: 1-94.

[0168] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence consists essentially of a sequence selected from the group consisting of SEQ ID NOs: 1-94.

[0169] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence is a sequence selected from the group consisting of SEQ ID NOs: 1-94.

[0170] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence consists of a sequence selected from the group consisting of SEQ ID NOs: 1-94.

[0171] In one embodiment of this aspect and all other aspects described herein, the nucleic acid sequence further comprises a trans-activating CRISPR RNA (tracrRNA) sequence.

[0172] In one embodiment of this aspect and all other aspects described herein, the nucleic acid molecule is a single guide RNA (sgRNA).

[0173] In one embodiment of this aspect and all other aspects described herein, the nucleic acid molecule comprises a vector.

[0174] In one embodiment of this aspect and all other aspects described herein, the vector is a viral vector, e.g., a lentiviral vector.

[0175] In one embodiment of this aspect and all other aspects described herein, the vector is an sgRNA expression vector.

[0176] In one embodiment of this aspect and all other aspects described herein, the method further comprises contacting the isolated progenitor cells with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease.

[0177] In one embodiment of this aspect and all other aspects described herein, the at least one DNA-targeting endonuclease is a Cas (CRISPR-associated) protein.

[0178] In one embodiment of this aspect and all other aspects described herein, the Cas protein is Cas9.

[0179] In one embodiment of this aspect and all other aspects described herein, the method further comprises providing an isolated cell, or an isolated progenitor cell, or an isolated population of cells, which can be progenitor cells or hematopoietic progenitor cells.

[0180] In one embodiment of this aspect and all other aspects described herein, the isolated cells are isolated progenitor cells.

[0181] In one embodiment of this aspect and all other aspects described herein, the isolated progenitor cells are isolated human cells.

[0182] In one embodiment of this aspect and all other aspects described herein, the isolated human cell is a hematopoietic progenitor cell or a hematopoietic stem cell. In other embodiments, the isolated human cell is an embryonic stem cell, a somatic stem cell, a progenitor cell, or a bone marrow cell.

[0183] In one embodiment of this aspect and all other aspects described herein, the methods described herein comprise contacting embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells or hematopoietic progenitor cells with an effective amount of a composition described herein or an effective amount of at least an isolated nucleic acid molecule described herein.

[0184] In another embodiment of this aspect and all other aspects described herein, the hematopoietic cells are erythroid cells. Methods for isolating hematopoietic progenitor cells are well known in the art, for example, by flow cytometry purification of CD34+ or CD133+ cells, microbeads conjugated with antibodies against CD34 or CD133, markers for hematopoietic progenitor cells. Commercially available kits are also available, for example, MACS® Technology CD34 MicroBead Kit, human, and CD34 MultiSort Kit, human, and STEMCELL™ Technology EasySep™ Mouse Hematopoietic Progenitor Cell Enrichment Kit.

[0185] In another embodiment of this aspect and all other aspects described herein, the hematopoietic stem cells, hematopoietic progenitor cells, embryonic stem cells, somatic stem cells, or progenitor cells are selected from peripheral blood, umbilical cord blood, chorionic villi, amniotic fluid, placental blood, or bone marrow.

[0186] In another embodiment of this aspect and all other aspects described herein, the human cell is an induced pluripotent stem cell (iPSC).

[0187] In another embodiment of this aspect and all other aspects described herein, the contacting of any cells described herein can be ex vivo or in vitro or in vivo.

[0188] In some embodiments of any of the methods or compositions described herein, the isolated progenitor cells or isolated cells are hematopoietic progenitor cells.

[0189] In some embodiments of any of the methods or compositions described herein, the hematopoietic precursors are cells of the erythroid lineage.

[0190] In some embodiments of any of the methods or compositions described herein, the isolated progenitor cells or isolated cells are induced pluripotent stem cells.

[0191] In another embodiment of this aspect and all other aspects described herein, the contacting of a cell of any of the aspects described herein comprises contacting the cell with an agent that binds to the cellular genomic DNA on chromosome 2 and causes an epigenetic modification in the cell's genome on chromosome 2, thereby reducing BCL11A mRNA or protein expression. In one embodiment, the epigenetic modification is on chromosome 2 at positions 60,716,189-60,728,612 (according to the UCSC Genome Browser hg 19 human genome assembly).

[0192] In one embodiment of this aspect and all other aspects described herein, the at least one epigenetic modification in the cellular genomic DNA on chromosome 2 indirectly or directly affects chromosome 2 positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region).

[0193] As used herein, "indirectly affecting chromosome 2 positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region)" refers to the long-range effects of epigenetic modifications in cellular genomic DNA on chromosome 2 positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region).

[0194] In another embodiment of this aspect and all other aspects described herein, the contacting of any cell described herein comprises contacting with an agent that binds to cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), 60722238-60722466 (+58 functional region), and / or 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly) and effects an epigenetic modification on chromosome 2, thereby reducing BCL11A mRNA or protein expression.

[0195] In another embodiment of this aspect and all other aspects described herein, contacting any cell described herein comprises contacting with an effective amount of a composition comprising at least one DNA targeting enzyme or a vector carrying a coding sequence for a DNA targeting enzyme, where the DNA targeting enzyme effects an epigenetic modification on chromosome 2, thereby reducing BCL11A mRNA or protein expression.

[0196] In another embodiment of this aspect and all other aspects described herein, the contacting of any cell described herein comprises contacting with an effective amount of a composition comprising at least one DNA-targeting enzyme or a vector carrying a coding sequence for a DNA-targeting enzyme, wherein the DNA-targeting enzyme effects an epigenetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly), thereby reducing BCL11A mRNA or protein expression. In one aspect, fetal hemoglobin expression is increased in the mammal compared to expression prior to the contacting.

[0197] In another embodiment of this aspect and all other aspects described herein, the hematopoietic progenitor cells, isolated human cells, or isolated cells are contacted ex vivo or in vitro.

[0198] In another embodiment of this aspect and all other aspects described herein, the at least one genetic modification is a deletion. In another embodiment of this aspect and all other aspects described herein, the at least one epigenetic modification.

[0199] In another embodiment of this aspect and all other aspects described herein, the deletion comprises one or more of DNAse 1-hypersensitive sites (DHS) +62, +58, and +55 as described herein in the Examples section. In another embodiment of this aspect and all other aspects described herein, the deletion consists essentially of one or more of DNAse 1-hypersensitive sites (DHS) +62, +58, and +55 as described herein in the Examples section. In another embodiment, the deletion consists of one or more of DNAse 1-hypersensitive sites (DHS) +62, +58, and +55 as described herein in the Examples section.

[0200] In another embodiment of this aspect and all other aspects described herein, the epigenetic modification comprises or affects one or more of DNAse 1-hypersensitive sites (DHS) +62, +58, and +55, as described herein in the Examples section. As used herein, the phrase "affecting one or more of DNAse 1-hypersensitive sites" means reducing the natural function of these DNAse 1-hypersensitive sites (DHS) +62, +58, and +55, for example, reducing their accessibility to transcription factors or DNA-degrading enzymes, such as DNase I. Generally, DNase I hypersensitive sites (DHS) are regions of chromatin that are sensitive to cleavage by DNase I enzymes. In these specific regions of the genome, chromatin loses its condensed structure, exposing and making the DNA accessible. This increases the availability of DNA for degradation by enzymes such as DNase I. These accessible chromatin zones are functionally related to transcriptional activity, as this remodeled state is required for the binding of proteins such as transcription factors. Thus, the epigenetic modifications contemplated herein reduce access to DNases by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold less, at least 2-fold less, at least 5-fold less, at least 10-fold less, at least 100-fold less, at least 1000-fold less, or more, compared to control cells not treated with any of the methods disclosed herein.

[0201] In another embodiment of this aspect and all other aspects described herein, the epigenetic modification is selected from the group consisting of 60,716,189 to 60,728,612, 60,716,189 to 60,723,870, 60,722,992 to 60,728,612, 60,717,236 to 60,719,036, 60,722,006 to 60,723,058, 60,724,917 to 60,726,282, 60,616,396 to 60,618,032 ...72 0,623,536 to 60,624,989, 60,626,565 to 60,628,177, 60,717,236 to 60,719,036, 60,721,212 to 60,722,958, 60,724,780 to 60,726,471, 60,739,075 to 60,740,154, 60,748,003 to 60,749,009, 60,826,438 to 60,827,601, or 60,831,589 to 60,833,556.

[0202] In another embodiment of this aspect and all other aspects described herein, the deletion removes the entire region of chromosome 2 at positions 60725424-60725688 (+55 functional region), 60722238-60722466 (+58 functional region), and / or 60718042-60718186 (+62 functional region), or removes part of the region, resulting in disruption of one or more DNAse 1-hypersensitive sites (DHS). As used herein, the term "interference" refers to a reduction in erythroid transcription of BCL11A in cells comprising interference with one or more DNAse-1 hypersensitive sites by at least 10% (e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or even 100% (i.e., no detectable erythroid transcription)) compared to cells without such interference. In one embodiment, interference comprises the inability of the modified DNAse-1 hypersensitive sites to bind to their native transcription factors (e.g., GATA1 and TAL1).

[0203] In another embodiment of this aspect and all other aspects described herein, the epigenetic modification that interferes with the establishment and / or maintenance of an epigenetic signature at an enhancer region on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly) thereby leads to reduced BCL11A mRNA or protein expression and reduces fetal hemoglobin expression in the mammal.

[0204] In one embodiment of this aspect and all other aspects described herein, epigenetic modifications that interfere with the establishment and / or maintenance of an epigenetic signature at an enhancer region on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly) include, but are not limited to, epigenetic modifications that affect DNase I sensitivity, epigenetic modifications that affect histone modifications, epigenetic modifications that affect GATA1 / TAL1 binding, and epigenetic modifications that affect long-range promoter interactions of the promoter of BCL11A.

[0205] For example, epigenetic modifications that interfere with the establishment and / or maintenance of epigenetic signatures at enhancer regions at the locations of the described functional regions on chromosome 2 include, but are not limited to, at least one deletion within positions 60,716,189-60,728,612 on chromosome 2, such that the overall function of this region is affected, thereby reducing or diminishing BCL11A mRNA and expression. For example, the deletion is located in a DNase I-sensitive region at positions 60,716,189-60,728,612 on chromosome 2, e.g., positions +62, +58, and +55. The deletion can also be located at positions +62, +58, or +55, or a combination thereof. For example, deletions can be located at positions +62 and +58, +58 and +55, +62 and +55, or all three positions +62, +58, and +55.

[0206] As another example, epigenetic modifications that interfere with the establishment and / or maintenance of an epigenetic signature at an enhancer region on the +55, +58, and +62 functional region of chromosome 2 include, but are not limited to, changes in histone modifications on chromosome 2 not at the location of the functional region, or changes in histone modifications on chromosome 2 at the location of the functional region, or changes in histone modifications on both chromosome 2 not at positions 60,716,189-60,728,612 and on chromosome 2 at positions 60,716,189-60,728,612, such that the overall function of this region is affected, thereby reducing or diminishing BCL11A mRNA and expression.

[0207] In another embodiment, the epigenetic modification that interferes with the establishment and / or maintenance of the epigenetic signature in the enhancer region at positions 60,716,189-60,728,612 on chromosome 2 includes, but is not limited to, the insertion of at least one modified specific suppressor sequence, which changes the epigenetic characteristics of non-coding elements in the functional regions of chromosome 2, +55, +58, and +62, thereby resulting in the suppression of target gene expression. The first method focuses specifically on epigenetically suppressing individual enhancers. In other words, the insertion of a modified specific suppressor sequence on chromosome 2 will initially interfere with the epigenetic modification in the BCL11A erythroid enhancer, which will ultimately lead to reduced BCL11A gene expression.

[0208] Any method known in the art can be used to effect the intended epigenetic modifications, for example, as described in Mendenhall EM et al., Nat. Biotechnol. 08 September 2013, and Maeder ML et al., Nat. Biotechnol. 09 October 2013.

[0209] In one embodiment of this aspect and all other aspects described herein, insertion of at least one modified specific suppressor sequence onto any location on chromosome 2 results in, but is not limited to, reduced DNase I sensitive regions at chromosome 2 location +55, +58, and +62 functional region; increased histone modifications at chromosome 2 location 60,716,189-60,728,612 or at the +55, +58, and +62 functional region; reduced transcription factor binding to GATA1 / TAL1 of the enhancer region on chromosome 2 location +55, +58, and +62 functional region; and reduced or weakened interaction between chromosome 2 location +55, +58, and +62 functional region and the BCL11A promoter.

[0210] In one embodiment of this aspect and all other aspects described herein, the overall effect of inserting at least one modified specific suppressor sequence onto any location on chromosome 2 is reduced or decreased BCL11A mRNA and expression.

[0211] In some embodiments, the term "reduced" or "diminished" when used in the context of BCL11A mRNA and expression, the +55, +58 and +62 functional regions on chromosome 2, or the interaction between the BCL11A enhancer and the BCL11A promoter, and transcription factor binding to GATA1 / TAL1 in the enhancer region, refers to at least 5% lower, at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 1-fold lower, at least 2-fold lower, at least 5-fold lower, at least 10-fold lower, at least 100-fold lower, at least 1000-fold lower, or more lower, compared to a control situation in which the epigenetic modification or insertion of the altered sequence disclosed herein is absent. Decreased BCL11A mRNA or protein expression in a cell means that protein expression is at least 5% lower, at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 1-fold lower, at least 2-fold lower, at least 5-fold lower, at least 10-fold lower, at least 100-fold lower, at least 1000-fold lower, or more lower, compared to a control cell that does not have the epigenetic modification or insertion of the altered sequence disclosed herein.

[0212] In one embodiment of this aspect and all other aspects described herein, the insertion of at least one modified specific suppressor sequence occurs within the DNase I sensitive region at positions 60,716,189-60,728,612 of chromosome 2, or at the +55, +58, and +62 functional regions. The insertion can also be at the 5' end of +62 or +58 or +55, or at the 3' end of +62 or +58 or +55, or between +62 and +58, or between +58 and +55, or between +55 and +62.

[0213] In one embodiment of this aspect and all other aspects described herein, the insertion of at least one modified specific suppressor sequence alters the DNase I sensitive region of the functional region at positions +55, +58 and +62 of chromosome 2.

[0214] In one embodiment of this aspect and all other aspects described herein, the epigenetic modification alters the DNase I sensitive region at positions 60,716,189 to 60,728,612 on chromosome 2 or the +55, +58 and +62 functional regions.

[0215] In one embodiment of this aspect and all other aspects described herein, the epigenetic modification alters a histone modification on chromosome 2 at positions 60,716,189 to 60,728,612, or at the +55, +58, and +62 functional regions.

[0216] In one embodiment of this aspect and all other aspects described herein, insertion of at least one modified specific suppressor sequence alters histone modifications on chromosome 2 at positions 60,716,189 to 60,728,612, or at the +55, +58, and +62 functional regions.

[0217] In one embodiment of this aspect and all other aspects described herein, the epigenetic modification alters GATA1 / TAL1 binding of the enhancer region on chromosome +55, +58, and +62 functional regions such that the overall function of this region is affected, thereby decreasing or reducing BCL11A mRNA and expression, e.g., transcription factor binding to GATA1 / TAL1.

[0218] In one embodiment of this aspect and all other aspects described herein, the insertion of at least one modified specific repressor sequence occurs within GATA1 / TAL1 as described herein. The insertion can be at the 5' or 3' end of GATA1 or TAL1. The insertion can be between GATA1 and TAL1. The insertion alters GATA1 / TAL1 binding to the enhancer region on chromosome 2 +55, +58, and +62 functional regions, such that the overall function of this region is affected, thereby lowering or decreasing BCL11A mRNA and expression. For example, the binding of transcription factors to GATA1 / TAL1.

[0219] In one embodiment of this and all other aspects described herein, the epigenetic modification alters the interaction between the BCL11A enhancer and the BCL11A promoter. In one embodiment, the interaction is reduced or attenuated such that the overall function of this region is affected, thereby reducing or decreasing BCL11A mRNA and expression.

[0220] In one embodiment of this aspect and all other aspects described herein, the epigenetic modification alters the interaction between the BCL11A promoter and the functional region at positions 60,716,189-60,728,612 and / or +55, +58, and +62 on chromosome 2. In one embodiment, the interaction is reduced or attenuated such that the overall function of this region is affected, thereby reducing or diminishing BCL11A mRNA and expression.

[0221] Also provided herein in another aspect are sequences encoding sequences from chromosome 2 at positions 60725424 to 60725688 (the +55 functional region), and / or at positions 60722238 to 60722466 (the +58 functional region), and / or at positions 60718042 to 60718186 (the +62 functional region) (UCSC Genome Browser hg 19 human genome assembly), wherein the genetically modified human cell has at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) or in BCL11A exon 2, wherein the DNA-targeting endonuclease cleaves the cellular genomic DNA, causing at least one genetic modification therein.

[0222] In another aspect, a gene encoding a gene encoding a gene encoding a gene for a mammal in need thereof, comprising a sequence encoding a gene encoding a gene for a mammal in need thereof, the sequence comprising a sequence ... 19 human genome assembly), wherein the at least one genetic modification is produced by a process of contacting the cell with an effective amount of a composition comprising at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) or at BCL11A exon 2, causing the at least one genetic modification therein.

[0223] In one embodiment of this aspect and all other aspects described herein, the isolated genetically modified human cell has at least one epigenetic modification in the cellular genomic DNA on chromosome 2. In another of this aspect and all other aspects described herein, the isolated genetically modified human cell has at least one epigenetic modification in the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) or in BCL11A exon 2.

[0224] In some aspects of any of these isolated genetically modified human cells having at least one epigenetic modification, the cells are transplanted into a mammal for use in increasing fetal hemoglobin in the mammal.

[0225] In one embodiment of this aspect and all other aspects described herein, the isolated genetically modified human cell, having at least one genetic modification in the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) or in BCL11A exon 2, is transplanted into a mammal for use in increasing fetal hemoglobin in the mammal.

[0226] In one embodiment of this aspect and all other aspects described herein, the isolated genetically modified human cells, having at least one genetic modification in the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) or in BCL11A exon 2, are stored by cryopreservation for later use.

[0227] In some aspects of any of the isolated genetically modified human cells having at least one epigenetic modification, the cells are stored for later use by cryopreservation.

[0228] In one embodiment of this aspect and all other aspects described herein, isolated genetically modified human cells having at least one genetic modification in the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) or in BCL11A exon 2 are cryopreserved, thawed, and transplanted into a mammal for use in increasing fetal hemoglobin in the mammal.

[0229] In some aspects of any of the isolated genetically modified human cells having at least one epigenetic modification, are cryopreserved, thawed, and transplanted into a mammal for use in increasing fetal hemoglobin in the mammal.

[0230] Another aspect provided herein relates to a composition comprising an isolated genetically modified human cell, wherein the cell has a sequence similar to that of chromosome 2 at positions 60725424-60725688 (+55 functional region), 60722238-60722466 (+58 functional region), and / or 60718042-60718186 (+62 functional region) (UCSC Genome Browser hg and / or positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly) created by a process of contacting cellular genomic DNA on chromosome 2 with an effective amount of a composition comprising a nucleic acid molecule or a vector carrying a nucleic acid molecule together with at least one DNA targeting endonuclease or a vector carrying a coding sequence for a DNA targeting endonuclease, wherein the DNA targeting endonuclease cleaves the cellular genomic DNA on chromosome 2 (according to the UCSC Genome Browser hg 19 human genome assembly) causing at least one genetic modification therein.

[0231] Another aspect provided herein relates to a composition comprising isolated genetically modified human cells for use in increasing fetal hemoglobin levels in a mammal in need thereof, wherein the cells are genetically modified to encode a gene encoding ... and / or positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly) created by a process of contacting the cell with an effective amount of a composition comprising a nucleic acid molecule or a vector carrying a nucleic acid molecule together with at least one DNA targeting endonuclease or a vector carrying a coding sequence for a DNA targeting endonuclease, wherein the DNA targeting endonuclease cleaves the cellular genomic DNA on chromosome 2 (according to the UCSC Genome Browser hg 19 human genome assembly) causing at least one genetic modification therein.

[0232] Another aspect provided herein relates to a composition comprising an isolated genetically modified human cell, wherein the cell has at least one epigenetic modification on chromosome 2. In one embodiment, the at least one epigenetic modification on chromosome 2 is at positions 60725424-60725688 (the +55 functional region), 60722238-60722466 (the +58 functional region), and / or 60718042-60718186 (the +62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly). In another embodiment, the at least one epigenetic modification on chromosome 2 is made by a process of contacting the cell with an effective amount of a composition comprising a nucleic acid molecule or a vector carrying a nucleic acid molecule together with at least one DNA targeting enzyme or a vector carrying a coding sequence for a DNA targeting enzyme, wherein the DNA targeting enzyme effects at least one epigenetic modification in the cellular genomic DNA on chromosome 2 affecting positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly).

[0233] Another aspect provided herein relates to a composition comprising an isolated genetically modified human cell for use in increasing fetal hemoglobin levels in a mammal in need thereof, wherein the cell has at least one epigenetic modification on chromosome 2. In one embodiment, the at least one epigenetic modification on chromosome 2 is at positions 60725424-60725688 (the +55 functional region), 60722238-60722466 (the +58 functional region), and / or 60718042-60718186 (the +62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly).

[0234] In one embodiment of this aspect and all other aspects described herein, the composition causes an increase in expression of fetal hemoglobin mRNA or protein in the contacted cells.

[0235] In one embodiment of this aspect and all other aspects described herein, the cells of any of the compositions described are autologous to the mammal that is the recipient of the cells in a transplant procedure, i.e., the cells of the composition are obtained or collected from the mammal prior to any of the described alterations or modifications.

[0236] In one embodiment of this aspect and all other aspects described herein, the cells of any of the compositions described are non-autologous to the mammal that is the recipient of the cells in a transplant procedure, i.e., the cells of the composition are not obtained or collected from the mammal prior to any of the described alterations or modifications.

[0237] In one embodiment of this aspect and all other aspects described herein, the cells of any composition described are, at a minimum, HLA-type matched to the mammal that is the recipient of the cells in a transplant procedure.

[0238] In one embodiment of this aspect and all other aspects described herein, the cells of any composition described are isolated progenitor cells prior to any alteration or modification described.

[0239] In one embodiment of this aspect and all other aspects described herein, the cells of any composition described are isolated hematopoietic progenitor cells prior to any alteration or modification described.

[0240] In one embodiment of this aspect and all other aspects described herein, the cells of any composition described are isolated induced pluripotent stem cells prior to any alteration or modification described.

[0241] In another embodiment of this aspect and all other aspects described herein, the deletion includes one or more of DNAse 1 hypersensitive sites (DHS) +62, +58, and +55 described herein in the Examples section. In another embodiment of this aspect and all other aspects described herein, the deletion consists essentially of one or more of DNAse 1 hypersensitive sites (DHS) +62, +58, and +55 described herein in the Examples section. In another embodiment, the deletion consists of one or more of DNAse 1 hypersensitive sites (DHS) +62, +58, and +55 described herein in the Examples section. In one embodiment, as used herein, the term "portion," in the context of a genomic deletion, refers to at least 10% to about 100% of a particular region. In another embodiment, the portion deleted is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or even 100% of the specified region.

[0242] In another embodiment of this aspect and all other aspects described herein, the deletion removes the entire region between positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) on chromosome 2 (according to the UCSC genome browser hg 19 human genome assembly), or removes a portion of the region, causing disruption of one or more DNAse 1 hypersensitive sites (DHS).

[0243] In one embodiment of this aspect and all other aspects described herein, the method further comprises selecting a mammal in need of increasing fetal hemoglobin.

[0244] In one embodiment of this aspect and all other aspects described herein, the mammal has been diagnosed with a hemoglobinopathy.

[0245] In one embodiment of this aspect and all other aspects described herein, the mammal in need of increasing fetal hemoglobin has been diagnosed with a hemoglobinopathy.

[0246] In one embodiment of this aspect and all other aspects described herein, the hemoglobinopathy is beta hemoglobinopathy.

[0247] In one embodiment of this aspect and all other aspects described herein, the hemoglobinopathy is sickle cell disease.

[0248] In one embodiment of this aspect and all other aspects described herein, the hemoglobinopathy is β-thalassemia.

[0249] In one embodiment of this aspect and all other aspects described herein, the contacted cells, human cells, hematopoietic progenitor cells or their progeny, are administered to a mammal.

[0250] In one embodiment, the present disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising: providing a population of hematopoietic progenitor cells or hematopoietic stem cells isolated from the mammal ex vivo, and encoding a cellular genomic DNA fragment on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region). and contacting a population of hematopoietic progenitor or stem cells with an effective amount of a composition comprising at least the nucleic acid molecule described herein or the vector described herein, together with a vector carrying a DNA-targeting endonuclease or a coding sequence for the DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves the NA, causing at least one genetic modification therein, thereby increasing the expression of fetal hemoglobin in the mammal compared to the expression before contacting. In a further embodiment of this method, the contacted population of hematopoietic progenitor or stem cells with increased fetal hemoglobin expression is cryopreserved, stored, or reintroduced into a mammal. In another embodiment, the cryopreserved population of hematopoietic progenitor or stem cells with increased fetal hemoglobin expression is thawed and then reintroduced into a mammal. In a further embodiment of this method, the method includes chemotherapy and / or radiation therapy to eliminate or reduce endogenous hematopoietic progenitor or stem cells in the mammal. In embodiments of any of the methods described, the hematopoietic progenitor or stem cells can be replaced with iPSCs as described herein.

[0251] In one embodiment, the present disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising: isolating a population of hematopoietic progenitor cells or hematopoietic stem cells from the mammal, and targeting the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region). and ex vivo contacting a population of hematopoietic progenitor or stem cells with an effective amount of a composition comprising at least the nucleic acid molecule or vector described herein, together with a DNA-targeting endonuclease or a vector carrying a coding sequence for the DNA-targeting endonuclease, whereby the targeting endonuclease cleaves and causes at least one genetic modification therein, thereby increasing the expression of fetal hemoglobin in the mammal compared to the expression before the contacting. In a further embodiment of this method, the ex vivo contacted population of hematopoietic progenitor or stem cells having increased fetal hemoglobin expression is cryopreserved, stored, or reintroduced into a mammal. In another embodiment, the cryopreserved population of hematopoietic progenitor or stem cells having increased fetal hemoglobin expression is thawed and then reintroduced into a mammal. In a further embodiment of this method, the method includes chemotherapy and / or radiation therapy to eliminate or reduce endogenous hematopoietic progenitor or stem cells in the mammal. In any of the embodiments of the methods described, the hematopoietic progenitor or stem cells can be replaced with iPSCs derived from a mammal. In any of the embodiments of the methods, the method further comprises selecting a mammal in need of increased fetal hemoglobin expression.

[0252] In one embodiment, the disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising: providing or isolating a population of hematopoietic progenitor or stem cells from the mammal; and deleting cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, thereby increasing expression of fetal hemoglobin in the mammal compared to expression before the contacting. In a further embodiment of this method, the population of hematopoietic progenitor or stem cells having the deleted genomic DNA and having increased fetal hemoglobin expression is cryopreserved, stored, or reintroduced into a mammal. In another embodiment, the population of hematopoietic progenitor or stem cells having deleted genomic DNA and increased fetal hemoglobin expression is thawed and then reintroduced into a mammal. In a further embodiment of this method, the method includes chemotherapy and / or radiation therapy to remove or reduce endogenous hematopoietic progenitor or stem cells in the mammal. In any of the described methods, the hematopoietic progenitor or stem cells can be replaced with iPSCs described herein. In any of the described methods, the hematopoietic progenitor or stem cells or iPSCs are similar to the mammal, meaning that the cells are derived from the same mammal. In another embodiment of the described methods, the hematopoietic progenitor or stem cells or iPSCs are dissimilar to the mammal, meaning that the cells are not derived from the same mammal, but from another mammal of the same species. For example, the mammal is a human. In any of the methods, the method further includes selecting a mammal in need of increased fetal hemoglobin expression.

[0253] In one embodiment, the present disclosure provides a method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising: isolating a population of hematopoietic progenitor or stem cells from the mammal, and deleting ex vivo cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, thereby increasing expression of fetal hemoglobin in the mammal compared to expression before the contacting. In a further embodiment of this method, the population of hematopoietic progenitor or stem cells having the deleted genomic DNA and having increased fetal hemoglobin expression is cryopreserved, stored, or reintroduced into a mammal. In another embodiment, the cryopreserved population of hematopoietic progenitor or stem cells having increased fetal hemoglobin expression is thawed and then reintroduced into a mammal. In a further embodiment of this method, the method includes chemotherapy and / or radiation therapy to remove or reduce endogenous hematopoietic progenitor or stem cells in the mammal. In any embodiment of the described methods, the hematopoietic progenitor or stem cells can be replaced with iPSCs derived from the mammal. In any embodiment of this method, the method further includes selecting a mammal in need of increased fetal hemoglobin expression.

[0254] In one embodiment of any of the methods described, the method further comprises selecting a mammal in need of increased fetal hemoglobin expression. Exemplary mammals in need of increased fetal hemoglobin expression are those diagnosed with a hemoglobinopathy.

[0255] In one aspect, the disclosure provides a method for treating a hemoglobinopathy in a mammal, comprising: (a) providing hematopoietic progenitor or stem cells or iPSCs; (b) contacting the cells ex vivo or in vitro with an effective amount of a composition comprising at least a nucleic acid molecule described herein or a vector described herein, together with a DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, where the DNA-targeting endonuclease cleaves the cellular genomic DNA at positions 60,716,189-60,728,612 on chromosome 2, causing at least one genetic modification therein, whereby expression of fetal hemoglobin is increased in the mammal compared to expression prior to the contacting; and (c) administering the cells of step (b) to the mammal.

[0256] In one embodiment of either method, the cells after step (b) can be cryopreserved until needed for administration to a mammal. In a further embodiment of this method, the method includes chemotherapy and / or radiation therapy to remove or reduce endogenous hematopoietic progenitor or stem cells in the mammal. In any embodiment of the described methods, the hematopoietic progenitor or stem cells or iPSCs are autologous to the mammal, meaning that the cells are derived from the same mammal. In another embodiment of the described methods, the hematopoietic progenitor or stem cells or iPSCs are non-autologous to the mammal, meaning that the cells are not derived from the same mammal, but from another mammal of the same species. For example, the mammal is a human.

[0257] In one embodiment of any of the methods described, the method further comprises selecting a mammal in need of treatment for a hemoglobinopathy.

[0258] In one embodiment, the disclosure provides a method for producing a hematopoietic progenitor or stem cell, comprising: (a) isolating hematopoietic progenitor or stem cells from a mammal; (b) contacting the cells ex vivo or in vitro with an effective amount of a composition comprising at least a nucleic acid molecule described herein or a vector described herein, together with a DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby expression of fetal hemoglobin is increased in the mammal compared to expression prior to the contacting; and (c) A method of treating a hemoglobinopathy in a mammal is provided, comprising administering to the mammal the cells of step (b).

[0259] In one embodiment, the cells after step (b) can be cryopreserved until needed for administration to a mammal. In any of the embodiments of this method, the method further comprises selecting a mammal in need of treatment for a hemoglobinopathy.

[0260] In one aspect, the disclosure provides a method for treating a hemoglobinopathy in a mammal, comprising: (a) providing hematopoietic progenitor or stem cells or iPSCs; (b) ex vivo deleting cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby expression of fetal hemoglobin is increased in the mammal compared to expression before the contacting; and (c) administering the cells of step (b) to the mammal.

[0261] In one embodiment, the cells after step (b) can be cryopreserved until needed for administration to a mammal. In a further embodiment of this method, the method includes chemotherapy and / or radiation therapy to remove or reduce endogenous hematopoietic progenitor or stem cells in the mammal. In any of the described methods, the hematopoietic progenitor or stem cells or iPSCs are similar to the mammal, meaning that the cells are derived from the same mammal. In another embodiment of the described methods, the hematopoietic progenitor or stem cells or iPSCs are dissimilar to the mammal, meaning that the cells are not derived from the same mammal, but from another mammal of the same species. For example, the mammal is a human. In any of the methods, the method further includes selecting a mammal in need of treatment for hemoglobinopathy.

[0262] In one aspect, the disclosure provides a method for treating a hemoglobinopathy in a mammal, comprising: (a) isolating hematopoietic progenitor or stem cells from a mammal; (b) ex vivo deleting cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region), causing at least one genetic modification therein, whereby expression of fetal hemoglobin is increased in the mammal compared to expression before the contacting; and (c) administering the cells of step (b) to the mammal.

[0263] In one embodiment, the cells after step (b) can be cryopreserved until needed for administration to a mammal. In a further embodiment of this method, the method includes chemotherapy and / or radiation therapy to remove or reduce endogenous hematopoietic progenitor or stem cells in the mammal. In any embodiment of this method, the method further includes selecting a mammal in need of treatment for a hemoglobinopathy.

[0264] In one embodiment, the present disclosure provides a method of treating a hemoglobinopathy in a mammal (e.g., a human), comprising introducing a composition described herein comprising an isolated, genetically engineered cell having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region), whereby expression of fetal hemoglobin is increased in the mammal. In a further embodiment of this method, the method comprises chemotherapy and / or radiation therapy to eliminate or reduce endogenous hemopoietic progenitor or stem cells in the mammal. In any of the embodiments of this method, the method further comprises selecting a mammal in need of hemoglobinopathy treatment.

[0265] In one embodiment, the present disclosure provides a method of treating a hemoglobinopathy in a mammal (e.g., a human) comprising increasing expression of fetal hemoglobin in the mammal by the methods described herein.

[0266] In one aspect of either method, the method further comprises selecting a subject diagnosed with or at risk for developing a hemoglobinopathy.

[0267] In one aspect of either method, the hemoglobinopathy is sickle cell disease (SCD) or thalassemia (THAL). For example, β-thalassemia.

[0268] In one aspect of the method, the method further comprises administering to the subject a treatment comprising oxygen, hydroxyurea, folic acid, or a blood transfusion.

[0269] In one aspect, the present disclosure provides a method of treating or reducing the risk of developing a hemoglobinopathy in a subject.

[0270] In any embodiment of any of the methods of treatment described, the hemoglobinopathy is beta hemoglobinopathy.

[0271] In any embodiment of any of the methods of treatment described, the hemoglobinopathy is β-thalassemia.

[0272] In any embodiment of any of the methods of treatment described, the hemoglobinopathy is sickle cell anemia.

[0273] In one embodiment of any of the methods described, the hematopoietic progenitor or stem cells or iPSCs are autologous to the mammal, meaning that the cells are derived from the same mammal. In another embodiment of any of the methods described, the hematopoietic progenitor or stem cells or iPSCs are non-autologous to the mammal, meaning that the cells are not derived from the same mammal, but from another mammal of the same species. For example, the mammal is a human.

[0274] In one embodiment of any of the methods described, the contacting of any of the cells described herein can be ex vivo or in vitro or in vivo.

[0275] In another embodiment of any of the methods described, contacting a cell of any of the methods described herein comprises contacting the cell with an agent that binds to cellular genomic DNA on chromosome 2 and results in an epigenetic modification in the cellular genome on chromosome 2, thereby reducing expression of BCL11A mRNA or protein. In one embodiment, the epigenetic modification is on chromosome 2 at positions 60725424-60725688 (the +55 functional region), and / or positions 60722238-60722466 (the +58 functional region), and / or positions 60718042-60718186 (the +62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly).

[0276] In another embodiment of any of the methods described, contacting any cell described herein comprises contacting with an agent that binds to the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly) and results in an epigenetic modification on chromosome 2, thereby reducing expression of BCL11A mRNA or protein.

[0277] In another embodiment of any of the methods described, contacting any of the cells described herein comprises contacting with an effective amount of a composition comprising at least a DNA-targeting enzyme or a vector carrying a coding sequence for a DNA-targeting enzyme that results in an epigenetic modification on chromosome 2, thereby reducing expression of BCL11A mRNA or protein.

[0278] In another embodiment of any of the methods described, contacting any of the cells described herein comprises contacting with an effective amount of a composition comprising at least a DNA-targeting enzyme or a vector carrying a coding sequence for a DNA-targeting enzyme that results in an epigenetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) (per the UCSC Genome Browser hg 19 human genome assembly), thereby reducing expression of BCL11A mRNA or protein. In one aspect, expression of fetal hemoglobin is increased in the mammal compared to expression before the contacting.

[0279] In another embodiment of any of the methods described, the hematopoietic progenitor cells, isolated human cells, or isolated cells are contacted ex vivo or in vitro.

[0280] In another embodiment of any method described, the at least one genetic modification is a deletion. In another embodiment of this aspect and all other aspects described herein, the at least one epigenetic modification.

[0281] In one aspect, provided herein is the use of an agent that binds to cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly) for increasing fetal hemoglobin in a mammal, or for treating a hemoglobinopathy in a mammal, or for reducing BCL11A mRNA or expression, wherein BCL11A mRNA or protein expression is reduced. In one embodiment, the agent is a nucleic acid molecule comprising a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region).

[0282] In one aspect, provided herein is an agent that binds to cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly) for use in a method for increasing fetal hemoglobin in a mammal, or for treating a hemoglobinopathy in a mammal, or for reducing BCL11A mRNA or expression, wherein BCL11A mRNA or protein expression is reduced. In one embodiment, the agent is a nucleic acid molecule comprising a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region).

[0283] In one embodiment, the present invention provides the use of an effective amount of a composition comprising the nucleic acid molecule described herein or the vector described herein in a method for increasing fetal hemoglobin in cells or mammals, or for treating hemoglobinopathy in mammals, or for reducing the mRNA or expression of BCL11A in cells or mammals.In one aspect, the method comprises transplanting cells with at least one epigenetic modification into a mammal, wherein the cells have been contacted with the composition described, comprising the nucleic acid described.

[0284] In one embodiment, the present invention provides a composition comprising the nucleic acid molecule described herein or the vector described herein, for use in a method for increasing fetal hemoglobin in cells or mammals, or for treating hemoglobinopathy in mammals, or for reducing the mRNA or expression of BCL11A in cells or mammals.In one aspect, the present invention provides a method comprising transplanting cells with at least one epigenetic modification into a mammal, wherein the cells have been contacted with the composition described, comprising the nucleic acid described.

[0285] A nucleic acid molecule comprising a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424 to 60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238 to 60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042 to 60718186 on human chromosome 2 (+62 functional region). In one embodiment, the composition further comprises at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), 60722238-60722466 (+58 functional region), and / or 60718042-60718186 (+62 functional region) to increase fetal hemoglobin in a mammal, or for treating a hemoglobinopathy in a mammal, or for reducing BCL11A mRNA or expression. In one aspect, a method comprising transplanting cells with at least one epigenetic modification into a mammal, wherein the cells have been contacted with a described composition comprising a described nucleic acid.

[0286] In one embodiment, provided herein is the use of an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting enzyme or a vector carrying a coding sequence for a DNA-targeting enzyme, the use being in a method for increasing fetal hemoglobin in a cell or mammal, or for treating a hemoglobinopathy in a mammal, or for reducing BCL11A mRNA or expression in a cell or mammal, wherein the DNA-targeting enzyme makes at least one epigenetic modification in cellular genomic DNA on chromosome 2, thereby affecting BCL11A mRNA or expression. In one embodiment, the at least one epigenetic modification is at positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region). In another embodiment, the effect of one epigenetic modification is to reduce BCL11A mRNA or protein expression. In one aspect, a method comprising transplanting cells with at least one epigenetic modification into a mammal.

[0287] In one aspect, provided herein is the use of an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein, said use being in a method for increasing fetal hemoglobin in a cell or mammal, or for treating a hemoglobinopathy in a mammal, or for reducing BCL11A mRNA or expression in a cell or mammal, wherein a DNA-targeting enzyme makes at least one epigenetic modification to cellular genomic DNA on chromosome 2, thereby affecting BCL11A mRNA or expression.

[0288] In one aspect, provided herein is a composition comprising a nucleic acid molecule described herein or a vector described herein for use in a method for increasing fetal hemoglobin in a cell or mammal, or for treating a hemoglobinopathy in a mammal, or for reducing BCL11A mRNA or expression in a cell or mammal, wherein a DNA-targeting enzyme makes at least one epigenetic modification to cellular genomic DNA on chromosome 2, thereby affecting BCL11A mRNA or expression.

[0289] In one embodiment, provided herein is a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting enzyme or a vector carrying a coding sequence for a DNA-targeting enzyme, for use in a method for increasing fetal hemoglobin in a cell or mammal, or for treating a hemoglobinopathy in a mammal, or for reducing BCL11A mRNA or expression in a cell or mammal, wherein the DNA-targeting enzyme makes at least one epigenetic modification in cellular genomic DNA on chromosome 2, thereby affecting BCL11A mRNA or expression. In one embodiment, the at least one epigenetic modification is at positions 60725424-60725688 (the +55 functional region), and / or at positions 60722238-60722466 (the +58 functional region), and / or at positions 60718042-60718186 (the +62 functional region). In another embodiment, the effect of one epigenetic modification is to reduce BCL11A mRNA or protein expression. In one aspect, a method comprising transplanting cells with at least one epigenetic modification into a mammal.

[0290] In one aspect, provided herein is the use of any of the isolated cells described herein in a method for increasing fetal hemoglobin in a mammal or for treating a hemoglobinopathy in a mammal. In one aspect, the method comprises transplanting the isolated modified cells described into a mammal.

[0291] In one aspect, provided herein is the use of a composition comprising isolated genetically modified human cells in a method of increasing fetal hemoglobin in a mammal or for the treatment of a hemoglobinopathy in a mammal, the composition comprising isolated genetically modified human cells, the cells comprising a gene encoding a gene encoding a gene encoding a gene for the hemoglobin-dependent phenotype of a mammal, the ... and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) on chromosome 2 (according to the UCSC Genome Browser hg 19 human genome assembly) created by the process of contacting the cell with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves the cellular genomic DNA on chromosome 2 (according to the UCSC Genome Browser hg 19 human genome assembly) causing at least one genetic modification therein. In one aspect, a method comprising transplanting into a mammal the described composition comprising the described isolated modified cell.

[0292] In one aspect, provided herein is a composition comprising an isolated genetically modified human cell for use in a method for increasing fetal hemoglobin in a mammal or treating a hemoglobinopathy in a mammal, the cell comprising a gene encoding a gene encoding a gene encoding a gene for a mammal, the ... and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) on chromosome 2 (according to the UCSC Genome Browser hg 19 human genome assembly) created by the process of contacting the cell with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves the cellular genomic DNA on chromosome 2 (according to the UCSC Genome Browser hg 19 human genome assembly) causing at least one genetic modification therein. In one aspect, a method comprising transplanting into a mammal the described composition comprising the described isolated modified cell.

[0293] In one aspect, provided herein is the use of a composition comprising an isolated genetically modified human cell in a method for increasing fetal hemoglobin in a mammal or for treating a hemoglobinopathy in a mammal, wherein the cell has at least one epigenetic modification on chromosome 2. In one aspect, the at least one epigenetic modification on chromosome 2 is at positions 60725424-60725688 (the +55 functional region), and / or positions 60722238-60722466 (the +58 functional region), and / or positions 60718042-60718186 (the +62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly). In another embodiment, the at least one epigenetic modification on chromosome 2 is produced by a process of contacting the cell with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA targeting enzyme or a vector carrying a coding sequence for a DNA targeting enzyme, wherein the DNA targeting enzyme effects at least one epigenetic modification in the cellular genomic DNA on chromosome 2 affecting positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly). In one aspect, a method comprising transplanting the described composition of isolated modified cells into a mammal.

[0294] In one embodiment, provided herein is a composition comprising an isolated genetically modified human cell for use in a method for increasing fetal hemoglobin in a mammal or for treating a hemoglobinopathy in a mammal, wherein the cell has at least one epigenetic modification on chromosome 2. In one embodiment, the at least one epigenetic modification on chromosome 2 is at positions 60725424-60725688 (the +55 functional region), and / or 60722238-60722466 (the +58 functional region), and / or 60718042-60718186 (the +62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly). In another embodiment, the at least one epigenetic modification on chromosome 2 is produced by a process of contacting the cell with an effective amount of a composition comprising a nucleic acid molecule described herein or a vector described herein together with at least one DNA targeting enzyme or a vector carrying a coding sequence for a DNA targeting enzyme, wherein the DNA targeting enzyme effects at least one epigenetic modification in the cellular genomic DNA on chromosome 2 affecting positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) (according to the UCSC Genome Browser hg 19 human genome assembly). In one aspect, a method comprising transplanting the described composition of isolated modified cells into a mammal.

[0295] In one aspect, provided herein is the use of any isolated cell described herein or any one of the compositions described herein for the manufacture of a medicament for use in increasing fetal hemoglobin in a mammal or for the treatment of a hemoglobinopathy in a mammal.

[0296] In one embodiment of the use of the compositions described herein, the composition causes an increase in fetal hemoglobin mRNA or protein expression in contacted cells.

[0297] In one aspect of the use of the compositions described herein, the cells of any of the compositions described are autologous to the mammal that is the recipient of the cells in a transplant procedure, i.e., the cells of the composition are derived from or taken from the mammal prior to any of the described modifications.

[0298] In one aspect of the use of the compositions described herein, the cells of any of the compositions described are non-autologous to the mammal that is the recipient of the cells in a transplant procedure, i.e., the cells of the composition are not derived from or taken from the mammal prior to any of the described modifications.

[0299] In one embodiment of the use of the compositions described herein, the cells of any of the compositions described are of a minimal HLA type compatible with the mammal that is the recipient of the cells in a transplant procedure.

[0300] In one embodiment of the use of the compositions described herein, the cells of any of the compositions described are isolated progenitor cells prior to any of the described modifications.

[0301] In one embodiment of the uses of the compositions described herein, the cells of any of the compositions described are isolated hematopoietic progenitor cells prior to any of the described modifications.

[0302] In one embodiment of the use of the compositions described herein, the cells of any of the compositions described are isolated induced pluripotent stem cells prior to any of the described modifications.

[0303] In one embodiment of the use of the compositions described herein, the cells of any of the compositions described are cryopreserved prior to use.

[0304] In one embodiment of any one of the methods described, the method is used to treat, prevent, or ameliorate a hemoglobinopathy, wherein the hemoglobinopathy is selected from the group consisting of hemoglobin C disease, sickle cell disease (SCD), sickle cell anemia, hereditary anemia, thalassemia, beta-thalassemia, thalassemia major, thalassemia intermedia, alpha-thalassemia, and hemoglobin H disease.

[0305] In various embodiments of any one of the methods described, the vector is administered in vivo by direct injection into cells, tissues, or organs of a subject requiring gene therapy. In various other embodiments of any one of the methods described, cells are transduced in vitro or ex vivo with a vector of the invention, and optionally expanded ex vivo. The transduced cells are then administered to a subject requiring gene therapy.

[0306] In one embodiment of any one of the methods described, the method further comprises selecting the subject who needs the gene therapy described.For example, the subject who shows symptoms or cytology of hemoglobinopathy is selected from the group consisting of hemoglobin C disease, hemoglobin sickle cell disease (SCD), sickle cell anemia, hereditary anemia, thalassemia, β-thalassemia, thalassemia major, thalassemia intermedia, α-thalassemia, and hemoglobin H disease.Alternatively, the subject has the gene mutation associated with hemoglobinopathy, which is the gene mutation described herein.For example, the subject who is diagnosed with SCD with genotype HbSS, HbS / β0 thalassemia, HbSD, or HbSO, and / or with HbF of <10% by electrophoresis.

[0307] In one aspect, the present disclosure provides a method of providing transduced or altered / genetically modified cells to a subject, comprising administering, e.g., parenterally administering, to a subject one or more cells transduced with a vector contemplated herein. In one embodiment, the vector is a vector carrying one or more nucleic acid sequences described herein; or is a nucleic acid molecule comprising a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, and wherein the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the entire genomic DNA sequence on human chromosome 2 at positions 60,716,189-60,728,612. In one embodiment, the nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1-94. In one embodiment, the nucleic acid molecule consists of a sequence selected from the group consisting of SEQ ID NOs: 1-94. In one embodiment, the nucleic acid molecule consists essentially of a sequence selected from the group consisting of SEQ ID NOs: 1-94.

[0308] In certain embodiments, methods are provided for preventing, ameliorating, or treating hemoglobinopathies in a subject. The methods comprise administering a population of cells comprising engineered / genetically modified hematopoietic stem or progenitor cells transduced therein with a vector contemplated herein. In one embodiment, the nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1-94. In one embodiment, the nucleic acid molecule consists of a sequence selected from the group consisting of SEQ ID NOs: 1-94. In one embodiment, the nucleic acid molecule consists essentially of a sequence selected from the group consisting of SEQ ID NOs: 1-94.

[0309] In certain embodiments of any of the methods described, the population of altered / genetically modified cells administered to the subject comprises hematopoietic stem or progenitor cells, proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, normochromatic erythroblasts, polychromatic erythrocytes, and red blood cells (RBCs), or any combination thereof, and any proportion that can be genetically modified by the vectors contemplated herein.

[0310] In some embodiments of any of the methods described, the population of engineered / genetically modified cells can be expanded in vitro or ex vivo in culture prior to implantation / engraftment into a subject or prior to cryopreservation for storage.

[0311] In some embodiments of any of the methods described, the population of engineered / genetically modified cells can be expanded in vitro or ex vivo in culture after cryopreservation and prior to implantation / engraftment into a subject.

[0312] In some embodiments of any of the methods described, the population of engineered / genetically modified cells can be differentiated in vitro or ex vivo prior to implantation into a subject.

[0313] The genetically modified cells may be administered as part of a bone marrow or umbilical cord blood transplant in individuals who have or have not undergone myeloablative therapy. In one embodiment, the genetically modified cells contemplated herein are administered in a bone marrow transplant to an individual who has undergone chemoablative or radioablative bone marrow therapy.

[0314] In one embodiment of any of the methods described, a dose of genetically modified cells is delivered to the subject intravenously. In one embodiment, genetically modified hematopoietic cells are administered intravenously to the subject.

[0315] In certain embodiments, the patient receives approximately 1 x 10 5 cells / kg, approximately 5×10 5 cells / kg, approximately 1×10 6cells / kg, approximately 2×10 6 cells / kg, approximately 3×10 6 cells / kg, approximately 4×10 6 cells / kg, approximately 5×10 6 cells / kg, approximately 6×10 6 cells / kg, approximately 7×10 6 cells / kg, approximately 8×10 6 cells / kg, approximately 9×10 6 cells / kg, approximately 1×10 7 cells / kg, approximately 5×10 7 cells / kg, approximately 1×10 8 In one embodiment, patients receive at least 1 x 10 cells / kg, or more, of the genetically modified cells, e.g., hematopoietic stem cells, in one single intravenous dose. 5 cells / kg, at least 5 × 10 5 cells / kg, at least 1 x 10 6 cells / kg, at least 2 x 10 6 cells / kg, at least 3 × 10 6 cells / kg, at least 4 × 10 6 cells / kg, at least 5 × 10 6 cells / kg, at least 6 × 10 6 cells / kg, at least 7 × 10 6 cells / kg, at least 8 × 10 6 cells / kg, at least 9 × 10 6 cells / kg, at least 1 x 10 7 cells / kg, at least 5 × 10 7 cells / kg, at least 1 x 10 8 cells / kg, or greater dose of genetically modified cells, e.g., hematopoietic stem cells described herein or genetically altered cells described herein or progeny thereof, in one single intravenous dose.

[0316] In additional embodiments, the patient receives about 1 x 10 5 cells / kg ~ approx. 1×10 8 cells / kg, approximately 1×10 6 cells / kg ~ approx. 1×10 8 cells / kg, approximately 1×10 6 cells / kg ~ approx. 9×10 6cells / kg, approximately 2×10 6 cells / kg ~ approx. 8×10 6 cells / kg, approximately 2×10 6 cells / kg ~ approx. 8×10 6 cells / kg, approximately 2×10 6 cells / kg ~ approx. 5×10 6 cells / kg, approximately 3×10 6 cells / kg ~ approx. 5×10 6 cells / kg, approximately 3×10 6 cells / kg ~ approx. 4×10 8 cells / kg dose, or any dose of cells / kg in between, of genetically modified cells, e.g., hematopoietic stem cells.

[0317] In various embodiments, the methods of the present invention provide more robust and safer gene therapy than existing methods and include administering to a subject a population or dose of cells comprising about 5% transduced / genetically modified cells, about 10% transduced / genetically modified cells, about 15% transduced / genetically modified cells, about 20% transduced / genetically modified cells, about 25% transduced / genetically modified cells, about 30% transduced / genetically modified cells, about 35% transduced / genetically modified cells, about 40% transduced / genetically modified cells, about 45% transduced / genetically modified cells, or about 50% transduced / genetically modified cells.

[0318] In one embodiment, the present invention provides genetically modified cells, e.g., stem cells, e.g., hematopoietic stem cells, that have the potential to expand or increase a population of red blood cells. In a particular embodiment, the hematopoietic stem cells are transduced with a vector of the present invention and administered to an individual in need of treatment for a hemoglobinopathy. Hematopoietic stem cells are of red blood cell origin and are therefore preferred. In one embodiment, the vector is a vector carrying one or more nucleic acid sequences described herein; or is a nucleic acid molecule comprising a nucleic acid sequence that is (a) complementary to the plus or minus strand of positions 60725424-60725688 on human chromosome 2 (+55 functional region); or (b) complementary to the plus or minus strand of positions 60722238-60722466 on human chromosome 2 (+58 functional region); or (c) complementary to the plus or minus strand of positions 60718042-60718186 on human chromosome 2 (+62 functional region), wherein human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, and wherein the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the entire genomic DNA sequence on human chromosome 2 at positions 60,716,189-60,728,612. In one embodiment, the nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1-94. In one embodiment, the nucleic acid molecule consists of a sequence selected from the group consisting of SEQ ID NOs: 1-94. In one embodiment, the nucleic acid molecule consists essentially of a sequence selected from the group consisting of SEQ ID NOs: 1-94.

[0319] In one embodiment, the genetically modified cell is further transduced with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region).

[0320] In one embodiment, the contacted hematopoietic stem cells described herein or genetically modified cells described herein or their progeny are implanted with prostaglandin E2 and / or the antioxidant N-acetyl-L-cysteine ​​(NAC) to promote engraftment of the respective cells.

[0321] In further embodiments of any of the methods described herein, the hematopoietic stem or progenitor cells that are contacted are erythroid cells.

[0322] In one embodiment of any of the methods described herein, the hematopoietic stem or progenitor cells are collected from peripheral blood, umbilical cord blood, chorionic villi, amniotic fluid, placental blood, or bone marrow.

[0323] In further embodiments of any of the methods described herein, the recipient subject is treated with chemotherapy and / or radiation prior to implantation of the contacted or transfected cells (i.e., the contacted hematopoietic stem cells described herein or the genetically modified cells described herein or their progeny).

[0324] In one embodiment, chemotherapy and / or radiation is to reduce endogenous stem cells to facilitate engraftment of the implanted cells.

[0325] In one aspect of either method, the contacted hematopoietic stem cells described herein or genetically modified cells described herein or their progeny are treated ex vivo with prostaglandin E2 and / or the antioxidant N-acetyl-L-cysteine ​​(NAC) to promote subsequent engraftment in the recipient subject.

[0326] Engraftment analysis was performed 4, 8, and 12 weeks after transplantation in peripheral blood and bone marrow, for example, by taking blood samples from these locations and determining BCL11A expression by any method known in the art.

[0327] In one aspect of any one of the methods described herein, the method includes obtaining a sample or population of embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells from a subject.

[0328] In one embodiment of any one of the methods described herein, the cell contacted with the nucleic acid molecule described herein, or the vector described herein, or the composition described herein comprising the nucleic acid molecule or vector is derived from an embryonic stem cell, a somatic stem cell, a progenitor cell, a bone marrow cell, a hematopoietic stem cell, or a hematopoietic progenitor cell.

[0329] In one embodiment, embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells are isolated from a host subject, transfected, cultured (optionally), and then re-transplanted into the same host (i.e., autologous cell transplantation). In another embodiment, embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells are isolated from a donor whose HLA type is matched with that of a host (recipient) diagnosed with or at risk of developing a hemoglobinopathy. Donor-recipient antigen type matching is well known in the art. HLA types include HLA-A, HLA-B, HLA-C, and HLA-D. These are the minimum cell surface antigen matches required for transplantation. That is, the transfected cells are transplanted into a different host (i.e., allogeneic to the recipient host subject). Donor or subject embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells can be transfected with a vector or nucleic acid containing the nucleic acid molecule described herein, and the transfected cells can be cultured and expanded, and then transplanted into a host subject. In one embodiment, the transplanted cells engraft into the host subject. Transfected cells can also be cryopreserved and stored after transfection, or cryopreserved and stored after cell expansion.

[0330] In one aspect of either method, the embryonic stem cells, somatic stem cells, progenitor cells, bone marrow cells, hematopoietic stem cells, or hematopoietic progenitor cells are autologous or allogeneic to the subject.

[0331] definition For convenience, certain terms employed throughout this application (including the specification, examples, and appended claims) are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0332] As used herein, the phrase "an agent that binds to cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region)" refers to a location within genomic DNA (e.g., chromosome 2 at positions 60725424-60725688 (+55 functional region), and "BCL11A-binding partner" refers to a small molecule, nucleic acid, protein, peptide, or oligonucleotide that can bind to positions 60722238-60722466 (the +58 functional region), and / or positions 60718042-60718186 (the +62 functional region) and that inhibits expression of BCL11A mRNA or protein in a cell by at least 20% compared to the BCL11A mRNA or protein levels in a cell that is not treated with such agent. In one embodiment, the agent "interferes with the interaction of BCL11A with a BCL11A-binding partner," as that term is used herein.

[0333] As used herein, the term "small molecule" refers to chemical compounds including, but not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds having a molecular weight of less than about 10,000 grams / mole (i.e., including heteroorganic compounds and organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 grams / mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams / mole, organic or inorganic compounds having a molecular weight of less than about 500 grams / mole, and salts, esters and other pharmaceutically acceptable forms of such compounds.

[0334] As used herein, a "nucleic acid" may be RNA or DNA, may be single-stranded or double-stranded, and may be selected from the group including, for example, a nucleic acid encoding a protein of interest, an oligonucleotide, a nucleic acid analogue, such as peptide nucleic acid (PNA), pseudocomplementary PNA (pc-PNA), locked nucleic acid (LNA), etc. Such nucleic acid sequences include, for example, but are not limited to, nucleic acid sequences encoding proteins that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, such as, but not limited to, RNAi, shRNAi, siRNA, microRNAi (mRNAi), antisense oligonucleotides, etc.

[0335] By "interfering with BCL11A interaction with a BCL11A binding partner" is meant that the amount of BCL11A interaction with a BCL11A binding partner is at least 5% lower in a population treated with a BCL11A inhibitor than in a comparable control population in which the BCL11A inhibitor is absent. Preferably, the amount of BCL11A interaction with a BCL11A binding partner in a population treated with a BCL11A inhibitor is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 1-fold lower, at least 2-fold lower, at least 5-fold lower, at least 10-fold lower, at least 100-fold lower, at least 1000-fold lower, or more. At a minimum, BCL11A interaction can be assayed by determining the amount of BCL11A binding to a BCL11A-binding partner using techniques standard in the art, including, but not limited to, mass spectrometry, immunoprecipitation, or gel filtration assays. Alternatively, or in addition, BCL11A activity can be assayed by measuring fetal hemoglobin expression at the mRNA or protein level after treatment with a candidate BCL11A inhibitor.

[0336] In one embodiment, BCL11A activity is the interaction of BCL11A with its binding partners: GATA-1, FOG-1, components of the NuRD complex, matrin-3, MTA2, and RBBP7. Therefore, any antibody or fragment thereof, small molecule, chemical substance, or compound that can block this interaction is considered an inhibitor of BCL11A activity.

[0337] As used herein, the term "genetically engineered cells," as that term is used herein, refers to cells that contain at least one genetic modification.

[0338] As used herein, the term " genetic modification " refers to the disruption at the genome level that causes the reduction of BCL11A expression or activity in cells.Exemplary genetic modifications can include deletion, frameshift mutation, point mutation, exon removal, removal of one or more DNAse 1 hypersensitive sites (DHS) (for example, two, three, four or more DHS regions), etc.

[0339] By "inhibiting BCL11A expression" is meant that the amount of BCL11A expression is at least 5% lower in cells or cell populations treated with a DNA-targeting endonuclease than in comparable control cells or cell populations in the absence of the DNA-targeting endonuclease. Preferably, the rate of BCL11A expression in the treated population is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 1-fold lower, at least 2-fold lower, at least 5-fold lower, at least 10-fold lower, at least 100-fold lower, at least 1000-fold lower, or more, than in a comparable control-treated population in which the DNA-targeting endonuclease is not added.

[0340] "Inhibiting BCL11A activity" means that the amount of BCL11A functional activity is at least 5% lower in cells or cell populations treated with the methods described herein than in comparable control cells or populations in which the DNA-targeting endonuclease is not present. Preferably, the rate of BCL11A activity in a population treated with a BCL11A inhibitor is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 1-fold lower, at least 2-fold lower, at least 5-fold lower, at least 10-fold lower, at least 100-fold lower, at least 1000-fold lower, or more than that of a comparable control-treated population in which the DNA-targeting endonuclease is not added. At a minimum, BCL11A activity can be assayed by determining the amount of BCL11A expression at the protein or mRNA level using standard techniques in the art. Alternatively, or in addition, BCL11A activity can be determined using a reporter construct that is sensitive to BCL11A activity. A sequence of the γ-globin locus can be recognized by a nucleic acid binding motif of the BCL11A construct.

[0341] In one embodiment, as used herein, the term "DNA-targeting endonuclease" refers to an endonuclease that generates a double-strand break at a desired location in the genome (e.g., positions 60,716,189-60,728,612 on chromosome 2) without generating undesired nonspecific double-strand breaks. The DNA-targeting endonuclease may be a naturally occurring endonuclease (e.g., a bacterial meganuclease) or may be artificially created (e.g., a genetically engineered meganuclease, TALEN, or ZFN, among others).

[0342] In another embodiment, as used herein, the term "DNA-targeting endonuclease" refers to an endonuclease that generates a single-strand break or a "nick" or cut in one strand of the DNA phosphate sugar backbone at a desired location in the genome (e.g., on chromosome 2, positions 60725424-60725688 (+55 functional region), and / or positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region)) without generating undesired non-specific DNA strand breaks.

[0343] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector, into which additional nucleic acid segments can be ligated into the viral genome. Some vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. Furthermore, some vectors can direct the expression of genes operably linked to them. Such vectors are referred to herein as "recombinant expression vectors," or more simply, "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Because plasmids are the most commonly used form of vector, "plasmid" and "vector" can be used interchangeably herein. However, the methods and compositions described herein can include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, lentiviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0344] Within the context of expression vectors, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to a regulatory sequence in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a target cell when the vector is introduced into the target cell). The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Furthermore, a DNA-targeting endonuclease can be delivered by a vector containing a regulatory sequence that directs the synthesis of the DNA-targeting endonuclease at specific intervals or for a specific period of time. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the target cell, the level of expression desired, and the like.

[0345] As used herein, the term "cleaving" generally refers to the creation of a double-stranded break in the DNA genome at a desired location.

[0346] As used herein, the term " effective amount of composition comprising at least DNA targeting endonuclease " refers to the amount of DNA targeting endonuclease that produces enough endonuclease activity to generate double-strand breaks at desired positions in genome.In one embodiment, the effective amount of DNA targeting endonuclease produces double-strand breaks at desired loci in at least 20% of the cells in the population that are contacted with the composition (for example, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or even 100% of the cells in the population comprise the genetic modification caused by the DNA targeting endonuclease composition).

[0347] As used herein, the term "increasing fetal hemoglobin levels" in a cell indicates that fetal hemoglobin is at least 5% higher in a population treated with an agent (e.g., a DNA-targeting endonuclease) that disrupts BCL11A mRNA or protein expression by binding to genomic DNA at positions 60,716,189-60,728,612 on chromosome 2 than in a comparable control population in the absence of the agent. Preferably, the rate of fetal hemoglobin expression in a population treated with such an agent that binds to genomic DNA at chromosome 2 positions 60,716,189-60,728,612 is at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 1-fold higher, at least 2-fold higher, at least 5-fold higher, at least 10-fold higher, at least 100-fold higher, at least 1000-fold higher, or more, than a control-treated population of comparable size and culture conditions. The term "control-treated population" is used herein to describe a population of cells treated with the same medium, viral induction, nucleic acid sequence, temperature, confluency, flask size, pH, etc., except for the addition of an agent that binds to genomic DNA at chromosome 2 positions 60,716,189-60,728,612. In one embodiment, an increase in fetal hemoglobin expression can be measured using any method known in the art, such as Western blot analysis of fetal gamma-globin protein and quantification of fetal gamma-globin mRNA.

[0348] As used herein, the term "isolated cell" refers to a cell that has been removed from an organism in which it is originally found, or the progeny of such a cell. Optionally, the cell has been cultured in vitro, for example, in the presence of other cells. Optionally, the cell is later introduced into a second organism or reintroduced into the organism from which the cell (or its descendant cells) was isolated.

[0349] As used herein, the term "isolated population" with respect to an isolated cell population refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, the isolated population is a substantially pure population of cells relative to the heterogeneous population from which the cells are isolated or enriched. In some embodiments, the isolated population is an isolated human hematopoietic progenitor cell population, e.g., a substantially pure human hematopoietic progenitor cell population relative to the heterogeneous population of cells comprising human hematopoietic progenitor cells and the cells from which the human hematopoietic progenitor cells are obtained.

[0350] The term "substantially pure" with respect to a particular cell population refers to a population of cells that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure relative to the cells that make up the entire cell population. In other words, the term "substantially pure" or "essentially purified" with respect to a population of hematopoietic progenitor cells refers to a population of cells that contains less than about 20%, more preferably less than about 15%, 10%, 8%, or 7%, and most preferably less than about 5%, 4%, 3%, 2%, or 1% or less than 1% of cells that are not hematopoietic progenitor cells as defined herein by this term.

[0351] As used herein, "subject" includes any animal exhibiting symptoms of a single-gene disease, disorder, or condition that can be treated with gene therapy vectors, cell-based therapies, and methods disclosed elsewhere herein. In preferred embodiments, the subject includes any animal exhibiting symptoms of a hematopoietic disease, disorder, or condition, e.g., a hemoglobinopathy, that can be treated with gene therapy vectors, cell-based therapies, and methods contemplated herein. Suitable subjects (e.g., patients) include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), livestock, and domestic animals or pets (e.g., cats or dogs). Included are non-human primates, preferably human patients. Typical subjects include animals that exhibit abnormal amounts (lower or higher than those of "normal" or "healthy" subjects) of one or more physiological activities that can be modulated by gene therapy.

[0352] In one embodiment, as used herein, "prevent" and similar terms, such as "prevented," "preventing," etc., refer to an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of a disease or condition. In another embodiment, the term refers to delaying the onset or recurrence of a disease or condition, or delaying the appearance or recurrence of symptoms of a disease or condition. In another embodiment, as used herein, "prevention" and similar terms include reducing the intensity, impact, symptoms, and / or burden of a disease or condition prior to the onset or recurrence of the disease or condition.

[0353] As used herein, the term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder. For example, the terms "treating" and "treatment" refer to administering an effective amount of a composition, e.g., an effective amount of a composition comprising a population of hematopoietic progenitor cells, to a subject such that the subject experiences a reduction in at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired clinical result. For purposes of this disclosure, a beneficial or desired clinical result includes, but is not limited to, alleviation of one or more symptoms, whether detectable or undetectable, a decrease in the extent of the disease, stabilization of the disease (e.g., not worsening), a delay or slowing of the progression of the disease, an improvement or palliation of the disease state, and remission (whether partial or total). In some embodiments, treating can refer to prolonging survival compared to expected survival in the absence of treatment. Thus, one of skill in the art will understand that treatment may improve a disease condition, but may not be a complete cure of the disease. In some embodiments, treatment can include prevention. However, in alternative embodiments, treatment does not include prevention.

[0354] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, and at a reasonable benefit / risk ratio.

[0355] As used herein, the terms "pharmaceutically acceptable," "physiologically acceptable," and grammatical variations thereof, when referring to compositions, carriers, diluents, and reagents, are used interchangeably to indicate that the material can be administered to or by a mammal without producing undesirable physiological effects, such as nausea, dizziness, upset stomach, and the like. A pharmaceutically acceptable carrier is not expected to promote the development of an immune response against the substance with which it is mixed, unless immunogenicity is desired. The preparation of pharmacological compositions containing active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on dosage form. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, although solid forms suitable for solution or suspension in liquid immediately prior to use can also be prepared. Preparations can also be emulsified or presented as liposomal compositions. The active ingredient can be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient, in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. Additionally, if desired, the composition may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient. The therapeutic compositions of the present invention may contain pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups can likewise be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Physiologically acceptable carriers are well known in the art.Exemplary liquid carriers are sterile aqueous solutions that contain no other materials than active ingredient and water, or contain both buffers such as sodium phosphate at physiological pH, such as saline or phosphate-buffered saline.Furthermore, water-soluble carriers can also contain two or more buffer salts, salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes.Liquid compositions can also contain liquid phases in addition to and excluding water.Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.The amount of active agent used in the methods described in this invention that is effective in treating specific disorders or conditions will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.

[0356] As used herein, " prevention " or " preventing " when used in relation to disease, disorder, or its symptoms refers to reducing the likelihood that an individual will develop a disease or disorder, such as hemoglobinopathy.The likelihood of developing a disease or disorder is reduced, for example, when an individual with one or more risk factors for a disease or disorder, compared with a group of individuals with the same risk factors but who have not undergone the treatment described herein, does not develop the disorder, or develops such a disease or disorder later or with less severity, statistically speaking.Not developing symptoms of the disease, or reducing (for example, at least 10% in the clinically accepted scale for the disease or disorder) or delaying (for example, by several days, weeks, months, or years) the onset of symptoms is considered to be effective prevention.

[0357] In the context of contacting cells with a DNA-targeting endonuclease to reduce BCL11A expression, the phrase "increasing fetal hemoglobin levels in cells" refers to fetal hemoglobin levels in cells or a population of cells treated with a DNA-targeting endonuclease being at least 5% higher than in a comparable control population in the absence of the DNA-targeting endonuclease. Preferably, fetal hemoglobin expression in cells treated with a DNA-targeting endonuclease is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold higher, at least 2-fold higher, at least 5-fold higher, at least 10-fold higher, at least 100-fold higher, at least 1000-fold higher, or more. The term "control-treated population" is used herein to describe a population of cells that has been treated with the same medium, viral induction, nucleic acid sequence, temperature, confluency, flask size, pH, etc., except for the addition of a BCL11A inhibitor.

[0358] The term "mammal" is intended to encompass the singular "mammal" and the plural "mammals," and includes, but is not limited to, humans; primates, such as apes, monkeys, orangutans, and chimpanzees; canines, such as dogs and wolves; felines, such as cats, lions, and tigers; equines, such as horses, donkeys, and zebras; food animals, such as cattle, pigs, and sheep; ungulates, such as deer and giraffes; rodents, such as mice, rats, hamsters, and guinea pigs; and bears. In some preferred embodiments, the mammal is a human.

[0359] Thus, in one embodiment, the mammal has been diagnosed with a hemoglobinopathy. In a further embodiment, the hemoglobinopathy is beta hemoglobinopathy. In one preferred embodiment, the hemoglobinopathy is sickle cell disease. As used herein, "sickle cell disease" can be sickle cell anemia, sickle hemoglobin C disease (HbSC), sickle beta-plus thalassemia (HbS / β+), or sickle beta-zero thalassemia (HbS / β0). In another preferred embodiment, the hemoglobinopathy is beta thalassemia.

[0360] As used herein, the term "hemoglobinopathy" refers to any defect in the structure or function of any hemoglobin in an individual, and includes defects in the primary, secondary, tertiary, or quaternary structure of hemoglobin caused by any mutation, such as a deletion or substitution mutation in the coding region of the β-globin gene, or a mutation or deletion in the promoter or enhancer of such a gene that results in a reduced amount of hemoglobin produced compared to normal or standard conditions. The term further includes any decrease in the amount or effectiveness of hemoglobin, whether normal or abnormal, caused by external factors such as disease, chemotherapy, toxins, poisons, etc.

[0361] In one embodiment, the term "effective amount" as used herein refers to a safe and sufficient amount of a cell composition to treat the onset of hemoglobinopathy, reduce the likelihood of hemoglobinopathy, or delay the onset of hemoglobinopathy. This amount can thus cure or improve hemoglobinopathy symptoms, slow the course of hemoglobinopathy disease progression, slow or prevent hemoglobinopathy symptoms, slow or prevent the establishment of secondary hemoglobinopathy symptoms, or prevent the onset of secondary hemoglobinopathy symptoms. The effective amount for treating hemoglobinopathy depends on the type of hemoglobinopathy being treated, the severity of the symptoms, the subject being treated, the age and general condition of the subject, the method of administration, and the like. Therefore, it is not possible or advisable to specify an exact "effective amount." However, for any given case, an appropriate "effective amount" may be determined by one of ordinary skill in the art using only routine experimentation.

[0362] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and each component thereof that is essential to the invention, accepting the inclusion of elements not specified, whether essential or not.

[0363] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristics of that embodiment of the invention.

[0364] The term "consisting of" refers to compositions, methods, and each component thereof described herein, excluding any element not recited in that description of an embodiment.

[0365] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods and / or steps of the type described herein that will become apparent to those of ordinary skill in the art upon reading this disclosure, and the like. It is understood that the foregoing detailed description and the following examples are merely illustrative and should not be construed as limitations on the scope of the invention. Various changes and modifications to the disclosed embodiments, which will be apparent to those skilled in the art, can be made without departing from the spirit and scope of the invention. Furthermore, all identified patents, patent applications, and publications are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodology described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the dates or representations as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the dates or contents of these documents.

[0366] Hemoglobinopathies Fetal hemoglobin (HbF) is a tetramer of two adult-type α-globin polypeptides and two fetal β-like γ-globin polypeptides. During pregnancy, the duplicated γ-globin gene constitutes the major gene transcribed from the β-globin locus. After birth, γ-globin is gradually replaced by adult-type β-globin, a process referred to as the "fetal switch" (3). The molecular mechanisms underlying this switch remain largely unclear and have been the subject of intensive research. The developmental switch from the production of primarily fetal hemoglobin, HbF (α2γ2), to adult hemoglobin, HbA (α2β2), begins around 28–34 weeks of gestation and continues until shortly after birth, when HbA becomes predominant. This switch is primarily driven by a decrease in γ-globin gene transcription and an increase in β-globin gene transcription. Normal adult blood contains, on average, only about 2% HbF, but residual HbF levels in healthy adults vary by more than 20-fold (Atweh, ​​Semin. Hematol. 38(4):367-73 (2001)).

[0367] Hemoglobinopathies include several inherited anemias characterized by reduced red blood cell (RBC) production and / or increased red blood cell (RBC) breakdown (hemolysis). These disorders also include genetic defects that result in the production of abnormal hemoglobin and a concomitant impairment of the body's ability to maintain oxygen levels. These disorders include the inability to produce sufficient amounts of normal beta-globin or the complete inability to produce normal beta-globin. These disorders, specifically those involving the beta-globin protein, are commonly referred to as beta-hemoglobinopathies. For example, beta-thalassemia results from partial or complete impairment of beta-globin gene expression, resulting in abnormal or absent hemoglobin A. Sickle cell anemia results from a point mutation in the beta-globin structural gene, resulting in the production of abnormal (sickle) hemoglobin (HbS). HbS RBCs are more fragile than normal RBCs and are more susceptible to hemolysis, ultimately leading to anemia (Atweh, ​​Semin. Hematol. 38(4):367-73 (2001)). Furthermore, the presence of a genetic variant in BCL11A, the HBS1L-MYB mutation, ameliorates the clinical severity of β-thalassemia. This variant has been shown to be associated with HbF levels. A high HbF variant has been shown to be associated with an odds ratio of 5 for having a less severe clinical form of β-thalassemia (Galanello S. et al., 2009, Blood, in press).

[0368] Pharmacological manipulation of fetal hemoglobin (α2γ2, HbF) has been a focus of therapeutic research aimed at reducing globin chain imbalance in patients with beta-hemoglobinopathies. The significant therapeutic potential of this approach is demonstrated by phenotypic observations in patients with both homozygous beta-thalassemia and hereditary hyperfetal hemoglobinemia (HPFH). Furthermore, the reduced transfusion requirement in patients with homozygous beta-thalassemia who lack adult hemoglobin synthesis is suggested by the presence of elevated fetal hemoglobin concentrations. Furthermore, some adult patients with beta-chain abnormalities have been found to have higher-than-normal fetal hemoglobin (HbF) concentrations and have been observed to have a milder disease course than patients with normal adult HbF levels. For example, a group of Saudi Arabian sickle cell disease patients with 20-30% HbF expression had mild clinical symptoms (Pembrey, et al., Br. J. Haematol. 40: 415-429 (1978)). It is now known that beta-hemoglobinopathies, such as sickle cell anemia and beta-thalassemia, can be improved by increasing HbF production (reviewed in Jane and Cunningham Br. J. Haematol. 102: 415-422 (1998) and Bunn, N. Engl. J. Med. 328: 129-131 (1993)).

[0369] The molecular mechanisms controlling the developmental in vivo switch from gamma globin to beta globin gene expression are currently unknown, but evidence is accumulating that external factors can affect gamma globin gene expression. The first group of compounds discovered to have HbF reactivation activity were cytotoxic drugs. It was first demonstrated in experimental animals using 5-azacytidine that pharmacological treatment could induce de novo synthesis of HbF (DeSimone, Proc Natl Acad Sci U S A. 79(14):4428-31 (1982)). Subsequent studies confirmed that 5-azacytidine can increase HbF in patients with beta thalassemia and sickle cell disease (Ley, et al., N. Engl. J. Medicine, 307: 1469-1475 (1982) and Ley, et al., Blood 62: 370-380 (1983)). Additional experiments demonstrated that baboons treated with cytotoxic doses of arabinosylcytosine (araC) responded with a significant increase in F-reticulocytes (Papayannopoulou et al., Science. 224(4649):617-9 (1984)), and that treatment with hydroxyurea led to the induction of gamma globin in monkeys or humans (Letvin et al., N Engl J Med. 310(14):869-73 (1984)).

[0370] The second group of compounds tested for their ability to induce HbF-reactivating activity were short-chain fatty acids. Early findings in fetal cord blood progenitor cells led to the discovery that gamma-aminobutyric acid could be a fetal hemoglobin inducer (Perrine et al., Biochem Biophys Res Commun. 148(2):694-700 (1987)). Subsequent studies showed that butyrate stimulates globin production in adult baboons (Constantoulakis et al., Blood. Dec; 72(6):1961-7 (1988)) and that it induces gamma-globin in erythroid cell lines from adult animals or patients with sickle cell anemia (Perrine et al., Blood. 74(1):454-9 (1989)). Derivatives of short-chain fatty acids, such as phenyl butyrate (Dover et al., Br J Haematol. 88(3):555-61 (1994)) and valproic acid (Liakopoulou et al., 1: Blood. 186(8):3227-35 (1995)), have also been shown to induce HbF in vivo. Given the large number of short-chain fatty acid analogs or derivatives in this family, several potential compounds in this family are more potent than butyrate. Phenylacetic acid and phenylalkyl acids (Torkelson et al., Blood Cells Mol Dis. 22(2):150-8 (1996)), discovered during subsequent research, belonged to this family of compounds and were therefore considered potential HbF inducers. However, currently, the use of butyrate or its analogs in sickle cell anemia and beta-thalassemia is still in the experimental stage and cannot be recommended for treatment outside of clinical trials.

[0371] Clinical trials aimed at reactivating fetal hemoglobin synthesis in sickle cell anemia and beta-thalassemia have included short- and long-term administration of compounds such as 5-azacytidine, hydroxyurea, recombinant human erythropoietin, and butyrate analogs, as well as combinations of these agents. Following these studies, hydroxyurea was used to induce HbF in humans and later became the first and final drug approved by the Food and Drug Administration (FDA) for the treatment of hemoglobinopathies. However, various drawbacks, including undesirable side effects and variable patient response, have contraindicated the long-term use of such agents or therapies. For example, hydroxyurea has been shown to stimulate HbF production and clinically alleviate sickling episodes, but is potentially limited by the risk of bone marrow toxicity and carcinogenesis. Potential long-term carcinogenicity may also exist with 5-azacytidine-based therapies. Erythropoietin-based therapies have not proven consistent across a range of patient populations. The short half-life of butyrate in vivo has been viewed as a potential obstacle to adapting these compounds for use in therapeutic intervention. Furthermore, very high doses of butyrate are required to induce γ-globin gene expression, necessitating catheterization for continuous infusion of the compound. Furthermore, these high doses of butyrate can be associated with neurotoxicity and multiorgan damage (Blau, et al., Blood 81: 529-537 (1993)). While even minimal increases in HbF levels are beneficial in sickle cell disease, β-thalassemia requires much higher increases that are not reliably or safely achieved by any currently available agent (Olivieri, Seminars in Hematology 33: 24-42 (1996)).

[0372] Identifying natural regulators of HbF induction and production may provide a means to devise therapeutic interventions that overcome the various drawbacks of the above-mentioned compounds. Recent genome-wide association studies have provided insight into the genetic basis of many complex diseases and traits (McCarthy et al., Nat Rev Genet 9, 356 (2008) and Manolio et al. J Clin Invest 118, 1590 (2008)). However, in the vast majority of cases, the functional link between the genetic association and the underlying pathophysiology remains unclear. Fetal hemoglobin (HbF) levels are inherited as a quantitative trait and are clinically important given their well-characterized role in ameliorating the severity of the major beta-hemoglobinopathies, sickle cell disease, and beta-thalassemia (Nathan et al., Nathan and Oski's Hematology of Infancy and Childhood ed. 6th, pp. 2 v. (xiv, 1864, xli p.) 2003). Two genome-wide association studies have identified three major genetic loci containing a set of five common single nucleotide polymorphisms (SNPs) that explain approximately 20% of the variance in HbF levels (Lettre et al., Proc Natl Acad Sci USA (2008); Uda et al., Proc Natl Acad Sci USA 105, 1620 (2008); Menzel et al., Nat Genet 39, 1197 (2007)). Furthermore, some of these variants appear to predict the clinical severity of sickle cell disease (Lettre et al., Proc Natl Acad Sci USA (2008)), and at least one of these SNPs may also influence the clinical outcome of beta-thalassemia (Uda et al., Proc Natl Acad Sci USA 105, 1620 (2008)). The SNP with the largest effect size, explaining more than 10% of the variance in HbF, is located in the second intron of the gene BCL11A on chromosome 2.The C2H2-type zinc finger transcription factor BCL11A has been investigated for its role in lymphocyte development (Liu et al., Nat Immunol 4, 525 (2003) and Liu et al., Mol Cancer 5, 18 (2006)), but its role in erythropoiesis or globin gene regulation has not previously been evaluated.

[0373] At the beginning of the recombinant DNA era, studies of globin gene structure provided a strong molecular basis for investigating the fetal globin switch. Considerable effort was devoted to delineating the cis-elements within the β-globin locus required for proper regulation of genes within the β-like globin cluster. These studies relied on naturally occurring mutations and deletions that dramatically affect adult HbF levels and were complemented by the generation of transgenic mice carrying portions of the cluster (Nathan et al., Nathan and Oski's Hematology of Infancy and Childhood ed. 6th, pp. 2 v. (xiv, 1864, xli p.) 2003) and G. Stamatoyannopoulos, Exp Hematol 33, 259 (2005)). Although the precise cis elements required for globin switching have not yet been clearly defined, findings in transgenic mice strongly suggest that the γ-globin genes are autonomously silenced in adulthood, a finding most compatible with the absence of fetal-specific activators or the presence of developmental stage-specific repressors. Recent genetic association studies have provided candidate genes, such as BCL11A, to investigate for their involvement in γ-globin gene regulation.

[0374] As used herein, treating or reducing the risk of developing hemoglobinopathy in a subject means improving at least one symptom of hemoglobinopathy. In one aspect, the present invention features a method for treating hemoglobinopathy in a subject, for example, reducing its severity or progression. In another aspect, the method can also be used to reduce the risk of developing hemoglobinopathy in a subject, delay the onset of hemoglobinopathy symptoms in a subject, or extend the lifespan of a subject with hemoglobinopathy. In one aspect, the method can include selecting a subject based on whether the subject has hemoglobinopathy, whether the subject is at risk of developing hemoglobinopathy but does not yet have it, or whether the subject has a latent hemoglobinopathy. Selecting a subject can include detecting symptoms of hemoglobinopathy, blood tests, genetic tests, or clinical records. If the test results indicate that the subject has a hemoglobinopathy, the method also includes administering a composition described herein to thereby treat or reduce the risk of developing the hemoglobinopathy in the subject, for example, a subject diagnosed with SCD with genotype HbSS, HbS / β0 thalassemia, HbSD, or HbSO, and / or with HbF of <10% by electrophoresis.

[0375] As used herein, the term "hemoglobinopathy" refers to a condition involving the presence of abnormal hemoglobin molecules in the blood. Examples of hemoglobinopathy include, but are not limited to, SCD and THAL. It also includes hemoglobinopathy in which a combination of abnormal hemoglobins is present in the blood (e.g., sickle cell / Hb-C disease). Typical examples of such diseases include, but are not limited to, SCD and THAL. SCD and THAL and their symptoms are well known in the art and are further described below. A subject can be diagnosed with hemoglobinopathy by a medical provider, medical caregiver, doctor, nurse, family member, or acquaintance who recognizes, understands, recognizes, determines, concludes, believes, or decides that the subject has a hemoglobinopathy.

[0376] The term "SCD" is defined herein to include any symptomatic anemic condition resulting from sickling of red blood cells. Symptoms of SCD include anemia; pain; and / or organ dysfunction, such as renal failure, retinopathy, acute chest syndrome, ischemia, priapism, and stroke. As used herein, the term "SCD" refers to various clinical problems associated with SCD, particularly in subjects homozygous for the sickle cell substitution in HbS. Among the constitutional symptoms referred to herein by the term "SCD" are delayed growth and development, an increased tendency to develop serious infections, particularly those caused by pneumococcus, marked impairment of splenic function, and the failure to effectively clear circulating bacteria, as well as recurrent infarction and eventual failure of splenic tissue. The term "SCD" also includes acute episodes of musculoskeletal pain, which primarily affect the lumbar spine, abdomen, and femoral shaft and are similar in mechanism and severity. In adults, these attacks typically manifest as brief, mild or moderate attacks every few weeks or months, interspersed with painful attacks lasting 5 to 7 days, occurring on average about once a year. Among the events known to trigger such crises are acidosis, hypoxia, and dehydration, all of which enhance the intracellular polymerization of HbS (J.H. Jandl, Blood: Textbook of Hematology, 2nd Ed., Little, Brown and Company, Boston, 1996, pages 544-545).

[0377] As used herein, "THAL" refers to a genetic disorder characterized by insufficient hemoglobin production. In one embodiment, the term encompasses hereditary anemia caused by mutations affecting hemoglobin synthesis. In another embodiment, the term encompasses any symptomatic anemia resulting from thalassemia conditions such as β-thalassemia major, thalassemia major, thalassemia intermedia, α-thalassemia, and hemoglobin H disease. β-thalassemia is caused by mutations in the β-globin chain and can occur as a major or minor form. In β-thalassemia major, children are normal at birth but develop anemia during the first year of life. β-thalassemia minor produces small red blood cells. Alpha-thalassemia is caused by the deletion of one or more genes from the globin chain.

[0378] The phrase "risk of developing a disease" refers to the relative probability that a subject will develop a hemoglobinopathy in the future compared to a control subject or population (e.g., a healthy subject or population). For example, an individual carrying a genetic mutation associated with SCD (an A to T mutation in the β-globin gene) increases the individual's risk, whether the individual is heterozygous or homozygous for the mutation.

[0379] Hematopoietic progenitor cells In one embodiment, the hematopoietic progenitor cells are contacted ex vivo or in vitro. In a specific embodiment, the contacted cells are erythroid lineage cells. In one embodiment, the cell composition comprises cells with reduced BCL11A expression.

[0380] "Hematopoietic progenitor cells," as the term is used herein, refer to cells of the stem cell lineage that give rise to all blood cell types, including the myeloid lineage (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid lineage (T cells, B cells, NK cells). "Cells of the erythroid lineage" indicates that the contacted cells are cells that undergo erythropoiesis, such as terminal differentiation to form erythrocytes or red blood cells (RBCs). Such cells belong to one of three cell lineages derived from bone marrow hematopoietic stem cells: erythroid, lymphoid, and myeloid. Upon exposure to specific growth factors and other components of the hematopoietic microenvironment, hematopoietic progenitor cells can mature into a series of intermediate differentiated cell types, all intermediates of the erythroid lineage, and then to RBCs. Thus, cells of the "erythroid lineage," as that term is used herein, include hematopoietic progenitor cells, proerythroblasts, basophil erythroblasts, erythroblasts, metaerythrocytes, reticulocytes, and erythrocytes.

[0381] In some embodiments, the hematopoietic progenitor cells have at least one of the following cell surface markers characteristic of hematopoietic progenitor cells: CD34+, CD59+, Thy1 / CD90+, CD38lo / -, and C-kit / CD117+. Preferably, the hematopoietic progenitor cells have several of these markers.

[0382] In some embodiments, the hematopoietic progenitor cells of the erythroid lineage have cell surface markers characteristic of the erythroid lineage: CD71 and Ter119.

[0383] Stem cells, such as hematopoietic progenitor cells, can proliferate and give rise to many more progenitor cells that have the potential to generate a large number of mother cells that can then give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and produce progeny that subsequently differentiate into one or more mature cell types while retaining one or more cells with the developmental potential of the parent. The term "stem cell" refers to a cell that, under certain circumstances, has the ability or potential to differentiate into a more specialized or differentiated phenotype and, under certain circumstances, retains the ability to proliferate without substantial differentiation. In one embodiment, the term progenitor cell or stem cell refers to a generalized mother cell whose progeny (progeny) specialize through differentiation, often in different directions, e.g., by acquiring completely individual characteristics, as occurs in the gradual diversification of embryonic cells and tissues. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells can be derived from pluripotent cells, which themselves are derived from pluripotent cells, etc. While each of these pluripotent cells can be considered a stem cell, the range of cell types each can give rise to can vary considerably. Some differentiated cells also have the ability to give rise to cells of greater developmental potential. Such ability can be natural or can be artificially induced upon treatment with various factors. In many biological cases, stem cells are also "pluripotent" because they can produce progeny of two or more distinct cell types, but this is not required for "stemness." Self-renewal is another classic part of the stem cell definition, which, as used herein, is essential. Theoretically, self-renewal can occur by either of two major mechanisms: stem cells can divide asymmetrically, with one daughter cell retaining the stem cell state and the other daughter cell expressing some distinct, other specific function and phenotype. Alternatively, some stem cells in a population can divide symmetrically into two stem cells, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise only to differentiated progeny.Generally, "progenitor cells" have a cell phenotype that is more primitive (i.e., earlier along a developmental pathway or progression than a fully differentiated cell). Progenitor cells often also have significant or very high proliferative potential. Depending on the developmental pathway and the environment in which the cell develops and differentiates, progenitor cells can give rise to multiple different differentiated cell types or to a single differentiated cell type.

[0384] In the context of cell ontogeny, the adjectives "differentiated" or "differentiating" are relative terms. A "differentiated cell" is a cell that is further along the developmental pathway than the cell to which it is being compared. Thus, stem cells can differentiate into lineage-committed progenitor cells (such as hematopoietic stem cells), which can then differentiate into other progenitor cell types further along the pathway (such as erythroid progenitors), and then into terminally differentiated cells, which may or may not play characteristic roles in certain tissue types and retain the ability to proliferate further.

[0385] induced pluripotent stem cells In some embodiments, the genetically engineered human cells described herein are derived from isolated pluripotent stem cells. An advantage of using iPSCs is that the cells can be derived from the same subject to which the progenitor cells are administered. That is, somatic cells can be obtained from a subject, reprogrammed into induced pluripotent stem cells, and then redifferentiated into hematopoietic progenitor cells (e.g., autologous cells) and administered to the subject. Because the progenitor cells are essentially derived from an autologous source, the risk of engraftment rejection or allergic reaction is reduced compared to using cells from another subject or group of subjects. In some embodiments, the hematopoietic progenitor cells are derived from a non-autologous source. In addition, the use of iPSCs eliminates the need to obtain cells from an embryonic source. Thus, in one embodiment, the stem cells used in the disclosed methods are not embryonic stem cells.

[0386] Although differentiation is generally irreversible under physiological conditions, several methods have been developed in recent years for reprogramming somatic cells into induced pluripotent stem cells. Exemplary methods are known in the art and are briefly described herein below.

[0387] As used herein, the term "reprogramming" refers to a process that changes or reverses the differentiation state of differentiated cells (e.g., somatic cells). In other words, reprogramming refers to a process that reverses the differentiation of cells toward more undifferentiated or more primitive cell types. It should be noted that many primary cultured cells can lose some of their fully differentiated characteristics when cultured. Thus, simple culturing of such cells, which are included in the term differentiated cells, does not result in these cells becoming non-differentiated (e.g., undifferentiated) or pluripotent cells. The transition of differentiated cells to pluripotency requires a reprogramming stimulus that exceeds the stimulus that leads to partial loss of differentiation characteristics in culture. Reprogrammed cells also have the characteristic of being able to be passaged for long periods of time without losing their proliferation potential, compared to primary cultured parent cells, which generally have the ability to divide a finite number of times in culture.

[0388] The cells to be reprogrammed can be partially differentiated or terminally differentiated before reprogramming. In some embodiments, reprogramming involves the complete reversal of the differentiation state of a differentiated cell (e.g., a somatic cell) to a pluripotent or multipotent state. In some embodiments, reprogramming involves the complete or partial reversal of the differentiation state of a differentiated cell (e.g., a somatic cell) to an undifferentiated cell (e.g., an embryonic-like cell). Reprogramming can result in the expression of specific genes by the cell, which contributes to further reprogramming. In certain embodiments described herein, reprogramming of a differentiated cell (e.g., a somatic cell) can cause the differentiated cell to assume an undifferentiated state (e.g., be an undifferentiated cell). The resulting cell is referred to as a "reprogrammed cell" or "induced pluripotent stem cell (iPSC or iPS cell)."

[0389] Reprogramming can involve at least some changes, e.g., reversal, of inherited patterns of nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cell differentiation. Reprogramming differs from simply maintaining the existing undifferentiated state of an already pluripotent cell, or from maintaining the existing, less-than-fully differentiated state of an already multipotent cell (e.g., hematopoietic stem cell). Reprogramming also differs from promoting self-renewal or proliferation of already pluripotent or multipotent cells, although the compositions and methods described herein can also be useful for such purposes in some embodiments.

[0390] The specific approach or method used to generate pluripotent stem cells from somatic cells (broadly referred to as "reprogramming") is not critical to the claimed invention. Thus, any method of reprogramming somatic cells to a pluripotent phenotype would be suitable for use in the methods described herein.

[0391] Reprogramming methodologies for generating pluripotent cells using defined combinations of transcription factors have been described in induced pluripotent stem cells. Yamanaka and Takahashi converted mouse somatic cells into ES cell-like cells with enhanced developmental potential by direct transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006). iPSCs resemble ES cells because they restore much of the pluripotency-associated transcriptional circuitry and epigenetic background. In addition, mouse iPSCs fulfill all standard assays for pluripotency, specifically in vitro differentiation into cell types of the three germ layers, teratoma formation, chimera commitment, germline transmission (Maherali and Hochedlinger, 2008), and tetraploid blastocyst complementation (Woltjen et al., 2009).

[0392] Subsequent experiments have shown that human iPS cells can be obtained using similar transduction methods (Lowry et al., 2008; Park et al., 2008; Takahashi et al., 2007; Yu et al., 2007b), and the transcription factor trio, OCT4, SOX2, and NANOG, have been established as a core set of transcription factors governing pluripotency (Jaenisch and Young, 2008). iPS cell production has historically been achieved by using viral vectors to introduce nucleic acid sequences encoding stem cell-associated genes into adult somatic cells.

[0393] iPS cells can be generated or derived from terminally differentiated somatic cells, as well as from adult somatic cells or somatic stem cells. That is, non-pluripotent progenitor cells can be reprogrammed to pluripotency or multipotency. In such instances, it is not necessary to include as many reprogramming factors as are required to reprogram terminally differentiated cells. Furthermore, reprogramming can be induced by introducing non-viral reprogramming factors, such as the protein itself, or by introducing a nucleic acid encoding the reprogramming factor, or by introducing a messenger RNA that produces the reprogramming factor upon translation (see, e.g., Warren et al., Cell Stem Cell, 2010 Nov 5;7(5):618-30). Reprogramming can be achieved by introducing a combination of nucleic acids encoding stem cell-related genes, including, for example, Oct-4 (also known as Oct-3 / 4 or Pouf51), Sox1, Sox2, Sox3, Sox15, Sox18, NANOG, Klf1, Klf2, Klf4, Klf5, NR5A2, c-Myc, l-Myc, n-Myc, Rem2, Tert, and LIN28. In one embodiment, reprogramming using the methods and compositions described herein can further include introducing one or more of Oct-3 / 4, Sox family members, Klf family members, and Myc family members into somatic cells. In one embodiment, the methods and compositions described herein further include introducing one or more of Oct4, Sox2, Nanog, c-MYC, and Klf4 for reprogramming. As noted above, the exact method used for reprogramming is not necessarily critical to the methods and compositions described herein. However, when cells differentiated from reprogrammed cells are to be used, for example, for human therapy, in one embodiment, the reprogramming is not done by methods that alter the genome.Thus, in such embodiments, reprogramming is performed without the use of, for example, viral or plasmid vectors.

[0394] The efficiency of reprogramming (i.e., the number of reprogrammed cells) derived from a starting cell population can be enhanced by the addition of various small molecules, as shown by Shi, Y., et al (2008) Cell-Stem Cell 2:525-528, Huangfu, D., et al (2008) Nature Biotechnology 26(7):795-797, and Marson, A., et al (2008) Cell-Stem Cell 3: 132-135. Thus, substances or combinations of substances that enhance the efficiency or rate of induced pluripotent stem cell production can be used to generate patient-specific or disease-specific iPSCs. Some non-limiting examples of substances that enhance reprogramming efficiency include soluble Wnt, Wnt-conditioned medium, BIX-01294 (G9a histone methyltransferase), PD0325901 (MEK inhibitor), DNA methyltransferase inhibitors, histone deacetylase (HDAC) inhibitors, valproic acid, 5'-azacytidine, dexamethasone, suberoylanilide, hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.

[0395] Other non-limiting examples of reprogramming enhancers include suberoylanilide hydroxamic acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamic acids), BML-210, depudecin (e.g., (-)-depudecin), HC toxin, nullscript (4-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide), phenylbutyrate (e.g., sodium phenylbutyrate), and valproic acid (( VPA) and other short-chain fatty acids), scriptaid, suramin sodium, trichostatin A (TSA), APHA compound 8, apicidin, sodium butyrate, pivaloyloxymethyl butyrate (Pivanex, AN-9), trapoxin B, chlamydocin, depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994 (e.g., N-acetyldinaline) and MS-27-275), MGCD0103, NVP- LAQ-824, CBHA (m-carboxycinnamic acid bishydroxamic acid), JNJ16241199, Tubacin, A-161906, proxamide, oxamflatin, 3-Cl-UCHA (for example, 6-(3-chlorophenylureido) caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Other reprogramming enhancers include, for example, dominant negative forms of HDAC (for example, catalytically inactive forms), siRNA inhibitors of HDAC, and antibodies that specifically bind to HDAC. Such inhibitors can be obtained from, for example, BIOMOL International, Fukasawa, Merck Biosciences, Novartis, Gloucester Pharmaceuticals, Aton Pharma, Titan Pharmaceuticals, Schering AG, Pharmion, MethylGene, and Sigma-Aldrich.

[0396] To confirm the induction of pluripotent stem cells for use with the methods described herein, isolated clones can be tested for the expression of stem cell markers. If stem cell markers are expressed in cells derived from somatic cells, the cells are identified as induced pluripotent stem cells. Stem cell markers can be selected from the non-limiting group including SSEA3, SSEA4, CD9, Nanog, Fbx15, Ecat1, Esg1, Eras, Gdf3, Fgf4, Cripto, Dax1, Zpf296, Slc2a3, Rex1, Utf1, and Nat1. In one embodiment, cells expressing Oct4 or Nanog are identified as pluripotent. Methods for detecting the expression of such markers can include, for example, RT-PCR and immunological methods that detect the presence of encoded polypeptides, such as Western blot or flow cytometry analysis. In some embodiments, detection not only involves RT-PCR but also includes the detection of protein markers. Intracellular markers can best be identified by RT-PCR, whereas cell surface markers are readily identified by, for example, immunocytochemistry.

[0397] The pluripotent stem cell characteristics of isolated cells can be confirmed by testing the differentiation ability of iPSCs into cells of each of the three germ layers. As an example, the pluripotent characteristics of isolated clones can be evaluated using teratoma formation in nude mice. The cells are introduced into nude mice, and histology and / or immunohistochemistry are performed on tumors arising from the cells. The development of tumors containing cells from all three germ layers, for example, further indicates that the cells are pluripotent stem cells.

[0398] Somatic Cells for Reprogramming As used herein, somatic cell refers to any cell that forms the body of an organism, except germline cell.All cell types in mammalian body are differentiated somatic cells, except sperm and egg, the cells from which sperm and egg are produced (gametocytes), and undifferentiated stem cells.For example, internal organs, skin, bone, blood, and connective tissue are all composed of differentiated somatic cells.

[0399] Additional somatic cell types for use with the compositions and methods described herein include fibroblasts (e.g., primary fibroblasts), muscle cells (e.g., muscle cells), cumulus cells, neural cells, mammary gland cells, hepatocytes, and pancreatic islet cells. In some embodiments, the somatic cells are primary cell lines or the progeny of primary or secondary cell lines. In some embodiments, the somatic cells are obtained from a human sample, such as a hair follicle, a blood sample, a biopsy (e.g., a skin biopsy, or an adipose tissue biopsy), a swab sample (e.g., a buccal swab), and are therefore human somatic cells.

[0400] Some non-limiting examples of differentiated somatic cells include, but are not limited to, epithelial cells, endothelial cells, neural cells, adipocytes, cardiac cells, skeletal muscle cells, immune cells, hepatocytes, splenocytes, lung cells, circulating blood cells, gastrointestinal cells, kidney cells, bone marrow cells, and pancreatic cells. In some embodiments, somatic cells can be primary culture cells isolated from any somatic tissue, including, but not limited to, brain, liver, gastrointestinal tract, stomach, intestinal tract, fat, muscle, uterus, skin, spleen, endocrine organs, bone, etc. Furthermore, somatic cells can be obtained from any mammalian species, non-limiting examples of which include murine, bovine, simian, porcine, equine, ovine, or human cells. In some embodiments, the somatic cells are human somatic cells.

[0401] When reprogrammed cells are used to generate hematopoietic progenitor cells for the therapeutic treatment of a disease, it is desirable, but not necessary, to use somatic cells isolated from the patient to be treated. For example, somatic cells associated with the disease or somatic cells involved in the therapeutic treatment of the disease can be used. In some embodiments, reprogrammed cells can be selected from a heterogeneous population containing reprogrammed cells and the somatic cells from which they are derived or generated by any known means. For example, reprogrammed cells can be isolated using a selectable marker, such as a drug resistance gene, as an indicator.

[0402] The reprogrammed somatic cells disclosed herein express genes encoding alkaline phosphatase (AP); ABCG2; stage-specific embryonic antigen-1 (SSEA-1); SSEA-3; SSEA-4; TRA-1-60; TRA-1-81; Tra-2-49 / 6E; ERas / ECAT5, E-cadherin; β-III-tubulin; α-smooth muscle actin (α-SMA); fibroblast growth factor 4 (Fgf4), Cripto, Dax1; zinc finger protein 296 (Zfp296); N-acetyltransferase-1 (Nat1); embryonic stem cell-associated transcript 1 (ECAT1); ESG1 / DPPA5 / ECAT2; ECAT3; ECAT6; ECAT7; ECAT 8; ECAT9; ECAT10; ECAT15-1; ECAT15-2; Fthl17; Sal14; Any number of pluripotent cell markers can be expressed, including blastocyst transcription factor (Utf1); Rex1; p53; G3PDH; telomerase including TERT; silent X chromosome genes; Dnmt3a; Dnmt3b; TRIM28; F-box-containing protein 15 (Fbx15); Nanog / ECAT4; Oct3 / 4; Sox2; Klf4; c-Myc; Esrrb; TDGF1; GABRB3; Zfp42, FoxD3; GDF3; CYP25A1; developmental pluripotency-associated 2 (DPPA2); T-cell lymphoma breakpoint 1 (Tcl1); DPPA3 / Stella; DPPA4; and other general markers for pluripotency. Other markers may include Dnmt3L; Sox15; Stat3; Grb2; β-catenin, and Bmi1. Such cells may also be characterized by the downregulation of markers characteristic of the somatic cells from which the induced pluripotent stem cells are derived.

[0403] Genome editing and DNA-targeting endonucleases As used herein, the term " genome editing " refers to the reverse genetic method, which uses artificially engineered nuclease to cut at desired position in genome and generate specific double-strand break, which is then repaired by the endogenous process of cell, such as homologous recombination (HR), homology-directed repair (HDR) and non-homologous end rejoining (NHEJ).NHEJ directly connects the DNA ends at double-strand break, while HDR uses homologous sequence as a template to regenerate the missing DNA sequence at break point.

[0404] Genome editing cannot be performed using conventional restriction endonucleases. Because most restriction enzymes recognize only a few base pairs on DNA as their targets, the recognized base pair combinations are likely to be found at many locations throughout the genome, resulting in multiple cuts (i.e., not limited to the desired location). To overcome this challenge and generate site-specific double-stranded breaks, several different classes of nucleases have been discovered and engineered to date. These include meganucleases, zinc finger nucleases (ZFNs), Cas9 / CRISPR systems, and transcription activator-like effector nucleases (TALENs).

[0405] Meganucleases are generally classified into four families: the LAGLIDADG family ("LAGLIDADG" disclosed in SEQ ID NO: 144), the GIY-YIG family, the His-Cys box family, and the HNH family. These families are characterized by structural motifs that affect catalytic activity and recognition sequences. For example, members of the LAGLIDADG family ("LAGLIDADG" disclosed in SEQ ID NO: 144) are characterized by having either one or two copies of the conserved LAGLIDADG motif (see Chevalier et al. (2001), Nucleic Acids Res. 29(18): 3757-3774). LAGLIDADG meganucleases ("LAGLIDADG" disclosed in SEQ ID NO: 144) with a single copy of the LAGLIDADG motif (SEQ ID NO: 144) form homodimers, while members with two copies of LAGLIDADG (SEQ ID NO: 144) are found as monomers. Similarly, GIY-YIG family members have a GIY-YIG module, which is 70-100 residues long and contains four or five conserved sequence motifs with four invariant residues, two of which are required for activity (see Van Roey et al. (2002), Nature Struct. Biol. 9: 806-811). His-Cys box meganucleases are characterized by a highly conserved series of histidines and cysteines over a region encompassing several hundred amino acid residues (see Chevalier et al. (2001), Nucleic Acids Res. 29(18): 3757-3774). In the case of the NHN family, its members are defined by a motif containing two pairs of conserved histidines surrounded by asparagine residues (see Chevalier et al. (2001), Nucleic Acids Res. 29(18): 3757-3774).The four families of meganucleases differ widely from each other with respect to conserved structural elements and, consequently, DNA recognition sequence specificity and catalytic activity.

[0406] Meganucleases are commonly found in microbial species and have very long recognition sequences (>14 bp), which makes them uniquely specific for cleavage at desired locations. This can be used to create site-specific double-strand breaks in genome editing. Those skilled in the art can utilize these natural meganucleases, but the number of such natural meganucleases is limited. To overcome this challenge, mutagenesis and high-throughput screening methods have been used to generate meganuclease variants that recognize unique sequences. For example, various meganucleases have been fused to generate hybrid enzymes that recognize new sequences. Alternatively, sequence-specific meganucleases can be designed by changing the DNA-interacting amino acids of meganucleases (see, for example, U.S. Patent No. 8,021,867). For example, meganucleases can be designed using the methods described in Certo, MT et al. Nature Methods (2012) 9:073-975; U.S. Patent No. 8,304,222; U.S. Patent No. 8,021,867; U.S. Patent No. 8,119,381; U.S. Patent No. 8,124,369; U.S. Patent No. 8,129,134; U.S. Patent No. 8,133,697; U.S. Patent No. 8,143,015; U.S. Patent No. 8,143,016; U.S. Patent No. 8,148,098; or U.S. Patent No. 8,163,514, the contents of each of which are incorporated herein by reference in their entirety. Alternatively, meganucleases with site-specific cleavage properties can be obtained using commercially available technology, for example, Precision BioScience's Directed Nuclease Editor™ genome editing technology.

[0407] ZFN and TALEN restriction endonuclease technologies utilize nonspecific DNA cleavage enzymes linked to specific DNA sequence-recognition peptides, such as zinc finger and transcription activator-like effectors (TALEs). Typically, an endonuclease is selected whose DNA recognition and cleavage sites are separate from each other, and the cleavage portion is then separated and linked to a sequence-recognition peptide, thereby obtaining an endonuclease with extremely high specificity for the desired sequence. An exemplary restriction enzyme with such properties is FokI. Furthermore, FokI has the advantage that it requires dimerization for nuclease activity, meaning that specificity increases dramatically as each nuclease partner recognizes a unique DNA sequence. To enhance this effect, FokI nucleases have been engineered to function only as heterodimers and have increased catalytic activity. Heterodimeric nucleases avoid the possibility of undesired homodimer activity, thus increasing the specificity of double-strand cleavage.

[0408] Although the nuclease moieties of both ZFNs and TALENs have similar properties, the difference between these engineered nucleases lies in their DNA recognition peptides. ZFNs rely on Cys2-His2 zinc fingers, while TALENs rely on TALEs. Both of these DNA-recognizing peptide domains are characterized by the fact that they are naturally found in combination within proteins. Cys2-His2 zinc fingers typically occur in repeat units spaced 3 bp apart and are found in various combinations in various nucleic acid-interacting proteins, such as transcription factors. TALEs, on the other hand, are found in repeat units with a 1:1 recognition ratio between the amino acid and the recognized nucleotide pair. Because both zinc fingers and TALEs occur in a repetitive pattern, different combinations can be attempted to produce a wide variety of sequence specificities. Approaches for generating site-specific zinc finger endonucleases include, for example, modular assembly (where zinc fingers associated with triplet sequences are attached in a row to cover the required sequence), OPEN (low-stringency selection of peptide domains vs. triplet nucleotides, followed by high-stringency selection of peptide combinations vs. final targets in bacterial systems), and bacterial one-hybrid screening of zinc finger libraries, among others. ZFNs for use in the methods and compositions described herein can be obtained commercially, for example, from Sangamo Biosciences™ (Richmond, CA).

[0409] It is contemplated herein that the Cas9 / CRISPR system of genome editing is utilized in the methods and compositions described herein.Clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system is useful for RNA programmable genome editing (see, for example, Jinek, M. et al. Science (2012) 337(6096):816-821).

[0410] Transactivating crRNA (tracrRNA) is a trans-encoded small RNA. It was first discovered in the human pathogen Streptococcus pyogenes (see Deltcheva E, et al. (2011). Nature 471 (7340):602-7). In bacteria and archaea, CRISPR / Cas (clustered regularly interspaced short palindromic repeats / CRISPR-associated proteins) constitutes an RNA-mediated defense system that protects against viruses and plasmids. This defense pathway has three steps. First, a copy of the invading nucleic acid is integrated into the CRISPR locus. Next, CRISPR RNA (crRNA) is transcribed from this CRISPR locus. The crRNA is then incorporated into an effector complex, where the crRNA guides the complex to the invading nucleic acid and the Cas protein degrades it (see Terns MP and Terns RM (2011). Curr Opin Microbiol 14 (3): 321-7). There are several pathways for CRISPR activation, one of which requires the tracrRNA, which plays a role in the maturation of the crRNA. The tracrRNA is complementary to and base-pairs with the pre-crRNA, forming an RNA duplex. This is cleaved by the RNA-specific ribonuclease RNase III to form a crRNA / tracrRNA hybrid. This hybrid acts as a guide for the endonuclease Cas9, which cleaves the invading nucleic acid (see Deltcheva E, et al. supra; Jinek M, et al. (2012), Science 337 (6096): 816-21; and Brouns SJ (2012), Science 337 (6096): 808-9).

[0411] Alternatively, genome editing can be performed using recombinant adeno-associated virus (rAAV)-based genome engineering, a genome editing platform centered on the use of rAAV vectors that allow for the insertion, deletion, or replacement of DNA sequences in the genome of living mammalian cells. The rAAV genome is a single-stranded deoxyribonucleic acid (ssDNA) molecule, either positive- or negative-sense, approximately 4.7 kilobases in length. These single-stranded DNA viral vectors have the unique property of high transduction rates and stimulating endogenous homologous recombination without causing double-stranded DNA breaks in the genome. Those skilled in the art can design rAAV vectors to target desired genomic loci and effect both global and / or subtle endogenous genetic changes in cells, such as deletions. rAAV genome editing has the advantage that it targets a single allele and does not cause any nonspecific genomic changes. rAAV genome editing technology is commercially available, for example, the rAAV GENESIS™ system from Horizon™ (Cambridge, UK).

[0412] Pharmaceutically acceptable carrier The methods of administering human hematopoietic progenitor cells or genetically modified cells, or their progeny, to a subject described herein involve using a therapeutic composition comprising hematopoietic progenitor cells. The therapeutic composition comprises a cell composition together with a physiologically acceptable carrier, and optionally at least one additional bioactive agent described herein dissolved or dispersed therein as an active ingredient. In preferred embodiments, the therapeutic composition is substantially non-immunogenic when administered to a mammal or human patient for therapeutic purposes, unless this is desired.

[0413] Generally, the hematopoietic progenitor cells or genetically modified cells described herein or their progeny are administered as a suspension with a pharmaceutically acceptable carrier.Those skilled in the art will recognize that the pharmaceutically acceptable carrier used in the cell composition does not contain a buffer, compound, cryoprotectant, preservative, or other agent in an amount that substantially interferes with the viability of the cells delivered to the subject.The cell-containing formulation may, for example, contain an osmotic buffer that can maintain the integrity of the cell membrane, and optionally nutrients to maintain the viability of the cells or enhance engraftment upon administration.Such formulations and suspensions are known to those skilled in the art and / or can be adapted for use with the hematopoietic progenitor cells described herein using routine experimentation.

[0414] The cell compositions may also be emulsified or presented as liposomal compositions, so long as the emulsification technique does not adversely affect the viability of the cells. The cells and any other active ingredients may be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients, in amounts suitable for use in the therapeutic methods described herein.

[0415] Additional agents contained in the cell compositions described herein may contain pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, tartaric acid, mandelic acid, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. Physiologically acceptable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions containing no materials other than the active ingredient and water, or containing both a buffer such as sodium phosphate at a physiological pH, such as saline or phosphate-buffered saline. Furthermore, aqueous carriers can contain two or more buffer salts, salts such as sodium chloride and potassium chloride, dextrose, polyethylene glycol, and other solutes. Liquid compositions can also contain liquid phases in addition to and excluding water. Examples of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of active compound used in the cell compositions described herein that will be effective in treating a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques.

[0416] In some embodiments, the compositions of isolated genetically modified cells described further comprise a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier does not comprise tissue or cell culture medium.

[0417] In some embodiments, the compositions of the described nucleic acid molecules further comprise a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier does not comprise tissue or cell culture medium.

[0418] In some embodiments, the compositions of vectors comprising the described nucleic acid molecules further comprise a pharmaceutically acceptable carrier. In one embodiment, the pharmaceutically acceptable carrier does not comprise tissue or cell culture medium.

[0419] Dosage and Indications As used herein, the terms "administer," "introduce," and "transplant" are used interchangeably in the context of placing cells, e.g., hematopoietic progenitor cells described herein, into a subject by a method or route that results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, to achieve a desired effect. Cells, e.g., hematopoietic progenitor cells, or their differentiated progeny, can be administered by any suitable route that results in delivery to a desired location in a subject where at least a portion of the implanted cells or cellular components remain viable. The survival period of the cells after administration to a subject can be as short as a few hours, e.g., 24 hours, to as long as several days or even years, i.e., long-term engraftment. For example, in some embodiments of the aspects described herein, an effective amount of hematopoietic progenitor cells or genetically modified cells with reduced BCL11A expression is administered by a systemic administration route, such as intraperitoneal or intravenous.

[0420] When provided prophylactically, the hematopoietic progenitor cells or genetically modified cells with reduced BCL11A expression described herein can be administered to a subject prior to any symptoms of hemoglobinopathy, for example, prior to the switch from fetal γ-globin to predominantly β-globin. Thus, prophylactic administration of a hematopoietic progenitor cell population is useful for preventing the hemoglobinopathy disclosed herein.

[0421] When provided therapeutically, the hematopoietic progenitor cells are provided at (or after) the onset of symptoms or signs of a hemoglobinopathy, eg, at the onset of sickle cell disease.

[0422] In some embodiments of the aspects described herein, the hematopoietic progenitor cell population or genetically modified cells with reduced BCL11A expression administered according to the methods described herein comprises allogeneic hematopoietic progenitor cells obtained from one or more donors.As used herein, "allogeneic" refers to hematopoietic progenitor cells or biological samples containing hematopoietic progenitor cells obtained from one or more different donors of the same species, where the genes at one or more loci are not identical.For example, the hematopoietic progenitor cell population or genetically modified cells with reduced BCL11A expression administered to a subject can be derived from umbilical cord blood obtained from one or more unrelated donor subjects or from one or more non-identical siblings.In some embodiments, a syngeneic hematopoietic progenitor cell population can be used, such as cells obtained from genetically identical animals or cells obtained from genetically identical twins.In other embodiments of this aspect, the hematopoietic progenitor cells are autologous cells. That is, hematopoietic progenitor cells are obtained or isolated from a subject and administered to the same subject, ie, the donor and recipient are the same.

[0423] For use in the various aspects described herein, an effective amount of hematopoietic progenitor cells or genetically modified cells with reduced BCL11A expression is at least 10 hematopoietic progenitor cells. 2 Cells, at least 5 x 10 2 cells, at least 10 3 Cells, at least 5 x 10 3 cells, at least 10 4 Cells, at least 5 x 10 4 cells, at least 10 5 Cells, at least 2 x 10 5 Cells, at least 3 x 10 5 Cells, at least 4 x 10 5 Cells, at least 5 x 10 5 Cells, at least 6 x 10 5 Hematopoietic progenitor cells, at least 7 x 10 5 Cells, at least 8 x 10 5 cells, at least 9 x 10 5 cells, at least 1 x 10 6Cells, at least 2 x 10 6 Cells, at least 3 x 10 6 Cells, at least 4 x 10 6 Cells, at least 5 x 10 6 Cells, at least 6 x 10 6 Cells, at least 7 x 10 6 Cells, at least 8 x 10 6 cells, at least 9 x 10 6 The hematopoietic progenitor cells or the genetically modified cells with reduced BCL11A expression can be derived from one or more donors, or can be obtained from autologous sources. In some embodiments of the aspects described herein, the hematopoietic progenitor cells are expanded in culture before being administered to a subject in need thereof.

[0424] In one embodiment, the term "effective amount" as used herein refers to the amount of a human hematopoietic progenitor cell population or its progeny required to alleviate at least one or more symptoms of a hemoglobinopathy, and relates to a sufficient amount of a composition to provide a desired effect, e.g., to treat a subject with a hemoglobinopathy. Thus, the term "therapeutically effective amount" refers to the amount of hematopoietic progenitor cells or genetically modified cells or their progeny, or a composition comprising hematopoietic progenitor cells or genetically modified cells or their progeny, that is sufficient to promote a particular effect when administered to a typical subject, such as a subject with or at risk for a hemoglobinopathy. As used herein, an effective amount also includes an amount sufficient to prevent or delay the onset of disease symptoms, alter the course of disease symptoms (e.g., but not limited to, slowing the progression of disease symptoms), or reverse disease symptoms. It is understood that for any given case, one of ordinary skill in the art can determine the appropriate "effective amount" using routine experimentation.

[0425] As used herein, "administered" refers to the delivery of a hematopoietic stem cell composition described herein into a subject by a method or route that results in at least partial localization of the cell composition at a desired site. The cell composition can be administered by any suitable route that results in effective treatment in the subject, i.e., administration results in delivery to a desired location in the subject, where at least a portion of the composition is delivered, i.e., at least 1 x 10 cells. 4 The cells are delivered to the desired site over a period of time. Methods of administration include injection, infusion, drip infusion, or ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intramedullary, intracerebrospinal, and intrasternal injection and infusion. For cell delivery, administration by injection or infusion is generally preferred.

[0426] In one embodiment, the cells described herein are administered systemically. As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" refer to administering a population of hematopoietic progenitor cells other than directly to a target site, tissue, or organ, such that the cells instead enter the subject's circulatory system and thus undergo metabolic and other similar processes.

[0427] A skilled clinician can determine the efficacy of treatment, including the compositions described herein for the treatment of hemoglobinopathy.However, if any one or all of the signs or symptoms of the disease, for example, fetal β-globin levels, are beneficially altered, and other clinically recognized symptoms or markers of the disease are improved or ameliorated by at least 10% after treatment with an inhibitor, the treatment is considered to be an "effective treatment," as this term is used herein.Efficacy can also be measured by the individual's lack of deterioration, as assessed by hospitalization, or the need for medical intervention (i.e., the progression of the disease is stopped or at least slowed).Methods for measuring these indicators are known to those skilled in the art and / or are described herein. Treatment includes any treatment of disease in an individual or animal (some non-limiting examples include humans or mammals), including (1) inhibiting the disease, e.g., halting or slowing the progression of sepsis; or (2) alleviating the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of developing infection or sepsis.

[0428] Treatment according to the present invention improves one or more symptoms associated with β-globin disorders by increasing the amount of fetal hemoglobin in an individual. Typical symptoms associated with hemoglobinopathies include, for example, anemia, tissue hypoxia, organ dysfunction, abnormal hematocrit, ineffective hematopoiesis, abnormal reticulocyte (red blood cell) count, abnormal iron loading, presence of ringed sideroblasts, splenomegaly, hepatomegaly, impaired peripheral blood flow, dyspnea, increased hemolysis, jaundice, anemic pain crises, acute chest syndrome, splenic hemocytosis, priapism, stroke, hand-foot syndrome, and pain, such as angina pectoris.

[0429] In one embodiment, hematopoietic progenitor cells are contacted with a DNA-targeting endonuclease ex vivo or in vitro, and the cells or their progeny are administered to a mammal (e.g., a human). In a further embodiment, the hematopoietic progenitor cells are erythroid lineage cells. In one embodiment, a composition comprising hematopoietic progenitor cells pre-contacted with a DNA-targeting endonuclease and a pharmaceutically acceptable carrier is administered to a mammal.

[0430] In one embodiment, an increase in fetal hemoglobin expression can be measured using any method known in the art, for example, Western blot analysis of fetal hemoglobin protein and quantification of fetal gamma-globin mRNA.

[0431] In one embodiment, hematopoietic progenitor cells are contacted with the DNA targeting endonuclease in vitro or ex vivo. In one embodiment, the cells are of human origin (e.g., autologous or heterologous). In one embodiment, the composition causes an increase in fetal hemoglobin expression.

[0432] The present invention may be defined in any of the following numbered paragraphs: [1] (a) complementary to the plus or minus strand of positions 60725424 to 60725688 (+55 functional region) of human chromosome 2; or (b) complementary to the plus or minus strand of positions 60722238 to 60722466 of human chromosome 2 (the +58 functional region); or (c) a nucleic acid sequence complementary to the plus or minus strand of positions 60718042 to 60718186 of human chromosome 2 (the +62 functional region); A nucleic acid molecule comprising: the human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly; the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the genomic DNA sequence at positions 60,716,189 to 60,728,612 on the human chromosome 2; Nucleic acid molecule. [2] The nucleic acid molecule of item 1, wherein the nucleic acid sequence excludes the entire BCL11A enhancer functional region. [3] The nucleic acid molecule of item 1, wherein the nucleic acid sequence excludes all of SEQ ID NOs: 136, 137, and 138. [4] The nucleic acid molecule of item 1, wherein the nucleic acid sequence is short and is 13 base pairs (bp) or longer. [5] The nucleic acid molecule of item 1, wherein the nucleic acid sequence is about 13 to 30 bp. [6] The nucleic acid molecule of item 1, wherein the nucleic acid sequence is about 20 bp. [7] The nucleic acid molecule of item 1, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1 to 94. [8] The nucleic acid molecule of item 1, wherein the nucleic acid sequence further comprises a trans-activating CRISPR RNA (tracrRNA) sequence. [9] The nucleic acid molecule of item 1, which is a single guide RNA (sgRNA).

[10] The nucleic acid molecule of item 1, including a vector.

[11] The nucleic acid molecule of item 10, wherein the vector is an sgRNA expression vector.

[12] (a) complementary to the plus or minus strand of positions 60725424 to 60725688 of human chromosome 2 (the +55 functional region); or (b) complementary to the plus or minus strand of positions 60722238 to 60722466 of human chromosome 2 (the +58 functional region); or (c) a nucleic acid sequence complementary to the plus or minus strand of positions 60718042 to 60718186 of human chromosome 2 (the +62 functional region); A vector comprising: the human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly; the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the genomic DNA sequence at positions 60,716,189 to 60,728,612 on the human chromosome 2; vector.

[13] The vector of item 12, wherein the nucleic acid sequence excludes the entire BCL11A enhancer functional region.

[14] The vector of item 12, wherein the nucleic acid sequence excludes the entirety of SEQ ID NOs: 136, 137, and 138.

[15] The vector of item 12, wherein the nucleic acid sequence is short and is 13 base pairs (bp) or longer.

[16] The vector of item 12, wherein the nucleic acid sequence is about 13 to 30 base pairs (bp).

[17] The vector of item 12, wherein the nucleic acid sequence is about 20 bp.

[18] The vector of item 12, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1-94.

[19] The vector of item 12, wherein the nucleic acid sequence further comprises a trans-activating CRISPR RNA (tracrRNA) sequence.

[20] The vector of item 12, which is an sgRNA expression vector.

[21] A method for producing progenitor cells having reduced BCL11A mRNA or protein expression, comprising contacting isolated progenitor cells with the nucleic acid molecule of any of items 1 to 11 or the vector of any of items 12 to 20.

[22] A method for producing progenitor cells with reduced BCL11A mRNA or BCL11A protein expression, comprising: contacting the isolated progenitor cells with an agent that binds to a human BCL11A enhancer functional region located on chromosome 2 at positions 60725424-60725688 (+55 functional region), 60722238-60722466 (+58 functional region), and / or 60718042-60718186 (+62 functional region); The agent is (a) the plus or minus strand of positions 60725424 to 60725688 (+55 functional region) of human chromosome 2; or (b) the plus or minus strand of positions 60722238 to 60722466 of human chromosome 2 (the +58 functional region); or (c) Positive or negative strand of human chromosome 2 at positions 60718042–60718186 (+62 functional region) binds to the human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly; reducing BCL11A mRNA or protein expression by said step. A method comprising:

[23] The method of item 21 or 22, further comprising contacting the isolated progenitor cells with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease.

[24] A method for producing an isolated genetically modified human cell having at least one genetic modification, comprising: contacting the isolated cells with an effective amount of a composition comprising the nucleic acid molecule of any of items 1 to 11 or the vector of any of items 12 to 20 together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, the DNA-targeting endonuclease cleaves the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) to cause at least one genetic modification therein. Including, Human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, Methods.

[25] The method of any of items 22 to 24, wherein the at least one DNA-targeting endonuclease is a Cas (CRISPR-associated) protein.

[26] The method of item 25, wherein the Cas protein is Cas9.

[27] The method of any of items 21 to 26, wherein the isolated progenitor cells or isolated cells are hematopoietic progenitor cells or hematopoietic stem cells.

[28] The method of item 27, wherein the hematopoietic precursors are erythroid cells.

[29] The method of any of items 21 to 26, wherein the isolated progenitor cells or isolated cells are induced pluripotent stem cells.

[30] The method of any of items 21 to 29, wherein the isolated progenitor cells or isolated cells are contacted ex vivo or in vitro.

[31] The method of any of items 21-30, wherein the contacted progenitor cell or the contacted cell acquires at least one genetic modification.

[32] The method of item 29, wherein at least one genetic modification is a deletion, insertion, or substitution of a nucleic acid sequence.

[33] Any of the methods of items 21 to 32, wherein at least one genetic modification is located on chromosome 2 at positions 60725424 to 60725688 (+55 functional region), and / or positions 60722238 to 60722466 (+58 functional region), and / or positions 60718042 to 60718186 (+62 functional region).

[34] The method of any of items 21 to 32, wherein the contacted progenitor cell or the contacted cell acquires at least one epigenetic modification in a BCL11A enhancer functional region.

[35] The method of item 34, wherein the at least one epigenetic modification is selected from the group consisting of DNA methylation, histone tail modifications, histone subunit organization, and nucleosome positioning alterations.

[36] The method of item 34 or 35, wherein at least one epigenetic modification is located on chromosome 2 at positions 60725424 to 60725688 (+55 functional region), and / or at positions 60722238 to 60722466 (+58 functional region), and / or at positions 60718042 to 60718186 (+62 functional region).

[37] An isolated, genetically modified human cell having at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), and / or at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) according to items 21-36.

[38] A composition comprising the isolated genetically modified human cell of item 37.

[39] A method for increasing fetal hemoglobin levels in a cell, comprising: contacting the isolated cells with an effective amount of a composition comprising the nucleic acid molecule of any of items 1 to 11 or the vector of any of items 12 to 20 together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) to cause at least one genetic modification therein; said step increasing fetal hemoglobin expression in said cell or its progeny compared to said cell prior to said contacting; Including, Human chromosome 2 from the UCSC Genome Browser hg 19 human genome assembly, method.

[40] The method of item 39, wherein the isolated cells are hematopoietic progenitor cells or hematopoietic stem cells.

[41] The method of items 39 or 40, wherein the hematopoietic progenitor cells are erythroid cells.

[42] The method of item 39, wherein the isolated cells are induced pluripotent stem cells.

[43] The method of any of items 39 to 42, wherein the isolated cells, hematopoietic progenitor cells, hematopoietic stem cells, or induced pluripotent stem cells are contacted ex vivo or in vitro.

[44] The method of any of items 39 to 43, wherein the at least one DNA-targeting endonuclease is a Cas (CRISPR-associated) protein.

[45] The method of item 44, wherein the Cas protein is Cas9.

[46] A method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising: contacting isolated hematopoietic progenitor cells in the mammal with an effective amount of a composition comprising the nucleic acid molecule of any of Items 1 to 11 or the vector of any of Items 12 to 20 together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) to cause at least one genetic modification therein; said step increasing fetal hemoglobin expression in said mammal compared to the expression before said contacting; Including, Human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly, Methods.

[47] A method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising transplanting the isolated genetically modified human cell of Item 37 or the composition of Item 38 into the mammal.

[0433] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, as well as figures and tables, cited throughout this application are hereby incorporated by reference. [Example]

[0434] Example 1 The present inventors have discovered and characterized regulatory elements of the BCL11A gene that are essential for its expression in erythroid cells. Common genetic variants within these sequences are associated with fetal hemoglobin levels and the severity of β-globin disorders. These sequences contain distal regulatory elements with enhancer chromatin signatures, which contain accessible chromatin, active histone marks, and occupancy by erythroid transcription factors. These elements interact with the BCL11A promoter and promote gene expression in erythroid cells but not in other cell lineages that express BCL11A, such as B lymphocytes. These regulatory elements can be targeted to therapeutically inhibit BCL11A and reinduce fetal hemoglobin. This can be achieved by mechanisms that include, but are not limited to, genome editing, nucleic acid or protein binding, and epigenetic modification. Advantages of this method include: disruption of physiological regulators of fetal hemoglobin levels, which cause increased γ-globin production and decreased β-globin production; minimal effects on overall globin production or on red blood cell formation or function; and limited effects on cells outside the erythroid lineage, thus reducing potential toxicity.

[0435] Enhancers are classically described as distal genetic elements that can positively regulate gene expression in an orientation-independent manner in ectopic gain-of-function expression experiments. 1 These elements regulate when, where, and how genes are expressed. Enhancer sequences bind transcription factors and chromatin regulators and correlate with specific chromatin features, including reduced DNA methylation, characteristic histone modifications, increased chromatin accessibility, long-range promoter interactions, and bidirectional transcription. Recent chromatin mapping has demonstrated that distal regulatory elements with enhancer chromatin signatures are enriched. 2-8 .

[0436] The biological importance of enhancers is underscored by gene expression studies that demonstrate the predictive power of enhancer profiles in lineage-specific programs. 9-12 Highly prominent and clustered enhancers (e.g., so-called strong enhancers, stretch enhancers, or super enhancers) are particularly indicative of cell identity and may help infer lineage-specific regulators. 13-15 Genome-wide association studies reveal enrichment of trait-associated variants in sequences with lineage-restricted enhancer signatures 7,13,16-19 Enhancers display signs of evolutionary repression and increased turnover, providing evidence of positive selection. 20-25 .

[0437] Regardless of their importance, enhancers are typically defined by criteria independent of in situ functional requirements. Advances in putative enhancer mapping, as well as large-scale oligonucleotide synthesis, facilitate enhancer reporter assays on a massively parallel scale, enabling systematic analysis of the functional significance of enhancer sequences. 26-30 Nevertheless, ectopic enhancer assays fail to address the necessity of elements in their native chromatin environment. The growing appreciation of the nonrandom distribution of distal elements both on the linear genome (e.g., into super-enhancer clusters) and within the three-dimensional nuclear environment highlights the importance of studying enhancers by perturbing their endogenous conditions. 15,31 .

[0438] Insightful observations were made by mutagenizing the enhancer using traditional molecular genetic approaches. 32,33 However, the low throughput of these classical methods limits their widespread application. Furthermore, the elevated turnover of many enhancer sequences between species may limit the ability to draw conclusions from non-human organisms regarding human gene regulation. Advances in genome editing technology make facile modification of the human genome practical. 34,35High-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 studies have revealed novel genes required for various biological processes. 36-41 Genome editing is similarly well suited to studying non-coding genetic elements such as enhancers, but these experiments have so far been performed at low throughput. 42-44 .

[0439] material and method Design and synthesis of human and mouse lentiviral sgRNA libraries. For every 20-mer sequence upstream of the NGG or NAG PAM sequence on the sense or antisense strand, orthologous +55, +58, and +62 DNase hypersensitive sites (DHSs) and BCL11A / Bcl11a exon 2 were identified in both humans and mice (Figures 6-11). We used references with the following substitutions near common low HbF-associated haplotypes relative to the human hg19 reference genome: rs1427407-G, rs1896293-T, rs6706648-T, rs6738440-G, and rs7606173-C. Each of the sgRNA oligos was synthesized as previously described. 37,41,64 The fragments were cloned into BsmBI-digested lentiGuide-Puro (Addgene plasmid ID 52963) using Gibson Assembly master mix (New England Biolabs), PCR-purified, and dephosphorylated. The Gibson Assembly products were transformed into electroporation-competent E. cloni® cells (Lucigen). Sufficient colonies were isolated to ensure approximately 90× library coverage for both the human and mouse libraries. Plasmid libraries were deep-sequenced (described below) to confirm representation.

[0440] To generate lentivirus, HEK293T cells were cultured in 15 cm tissue-culture-treated Petri dishes in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) supplemented with 10% fetal bovine serum (FBS) (Omega Scientific) and 2% penicillin-streptomycin (Life Technologies). HEK293T cells were transfected at 80% confluence with 13.3 μg of psPAX2, 6.7 μg of VSV-G, and 20 μg of the lentiviral construct plasmid of interest in 12 mL of medium using 180 μg of branched polyethylenimine (Sigma). 16–24 h after transfection, the medium was changed. Lentiviral supernatants were collected at 48 and 72 h posttransfection and then concentrated by ultracentrifugation (24,000 rpm, 4°C, 2 h using a Beckman Coulter SW 32 Ti rotor).

[0441] A tiled pooled CRISPR-Cas9 screen for in situ functional mapping of the human BCL11A erythroid enhancer. HUDEP clone 2 (HUDEP-2) was utilized as previously described by Nakamura and coworkers. 49 HUDEP-2 cells were cultured at 10 -6 Cells were grown in StemSpan SFEM (Stem Cell Technologies) supplemented with 1 μg / mL dexamethasone (Sigma), 100 ng / mL human stem cell factor (SCF) (R&D), 3 IU / mL erythropoietin (Amgen), 1% L-glutamine (Life Technologies), and 2% penicillin / streptomycin (Life Technologies). Cultures were supplemented with 1 μg / mL doxycycline (Sigma) to induce expression of the human papillomavirus type 16 E6 / E7 gene. 49HUDEP-2 cells were differentiated in Iscove's modified Dulbecco's medium (IMDM) supplemented with 330 μg / mL holotransferrin (Sigma), 10 μg / mL recombinant human insulin (Sigma), 2 IU / mL heparin (Sigma), 5% human solvent detergent pooled plasma AB (Rhode Island Blood Center), 3 IU / mL erythropoietin (Amgen), 100 ng / mL human stem cell factor (SCF) (R&D), 1 μg / mL doxycycline (Sigma), 1% L-glutamine (Life Technologies), and 2% penicillin / streptomycin (Life Technologies).

[0442] HUDEP-2 cells with stable Cas9 expression were transduced with a human sgRNA library lentiviral pool at low multiplicity in Expansion Medium. Control transductions were performed to ensure the transduction rate did not exceed 50%. Cell numbers were maintained at appropriate levels exceeding 1000x library representation throughout the experiment. 24 hours after transduction, 10 μg / mL blasticidin (Sigma) and 1 μg / mL puromycin (Sigma) were added to select for lentiviral library components in the cells with Cas9. Cells were cultured in Expansion Medium for 1 week, followed by an additional week in Differentiation Medium.

[0443] Intracellular staining was performed by fixing cells with 0.05% glutaraldehyde (grade II) (Sigma) for 10 minutes at room temperature. Cells were centrifuged at 350g for 5 minutes and then resuspended in 0.1% Triton-X 100 (Life Technologies) for 5 minutes at room temperature for permeabilization. Triton X-100 was diluted with phosphate-buffered saline (PBS) and then centrifuged at 350g for 15 minutes. Cells were stained with anti-human antibody against HbF (clone HbF-1 with FITC or APC conjugation; Life Technologies) and β-hemoglobin antibody (clone 37-8 with PerCP-Cy5 or PE conjugation; Santa Cruz) for 20 minutes in the dark. Before FACS analysis, cells were washed to remove unbound antibody. 0.2 μg of HbF antibody and 2 μg of HbA (β-hemoglobin) antibody were used per 5 million cells. Non-targeting sgRNA samples and control cells exposed to BCL11A exon 2 were used as negative and positive controls, respectively, to establish flow cytometry conditions. Cell populations within 10% above and below HbF expression were sorted by FACS.

[0444] After sorting the high and low HbF pools, library preparation and deep sequencing were performed as previously described. 37Briefly, genomic DNA was extracted using the Qiagen Blood and Tissue kit. Herculase PCR reactions (Agilent) using lentiGuide-Puro-specific primers containing the handle sequence were performed as follows: Herculase II reaction buffer (1x), forward and reverse primers (0.5 μM each), dimethyl sulfoxide (DMSO) (8%), deoxynucleotide triphosphates (dNTPs) (0.25 mM each), and Herculase II Fusion DNA Polymerase (0.5 reactions). The following cycling conditions were used: 95°C for 2 minutes; 20 cycles of 95°C for 15 seconds, 60°C for 20 seconds, and 72°C for 30 seconds; and 72°C for 5 minutes. Amplification was performed from 6.6 μg of gDNA (approximately 10e6 cell genomes) per pool using multiplex reactions of up to 200 ng each. Samples were subjected to a second PCR using handle-specific primers to add adapters and indexes to each sample. The following conditions were used: Herculase II reaction buffer (1x), forward and reverse primers (0.5 μM each), deoxynucleotide triphosphates (dNTPs) (0.25 mM each), and Herculase II Fusion DNA Polymerase (0.5 reactions) with the following cycling conditions: 95°C for 2 minutes; 25 cycles of 95°C for 15 seconds, 60°C for 20 seconds, and 72°C for 30 seconds; 72°C for 5 minutes. PCR products were run on an agarose gel, and bands of the expected size were gel-purified. Illumina MiSeq 150-bp paired-end sequencing was performed.

[0445] The sgRNA sequences present in the plasmid pool and the high and low HbF pools were listed. Reads were normalized to the sequencing depth per library. The extinction score was determined by calculating (1) the ratio of normalized reads in the high HbF pool compared to the low HbF pool; (2) log2 transformation; and (3) the median of biological replicates. The HbF enrichment score was determined by calculating (1) the ratio of normalized reads in the high HbF pool compared to the low HbF pool; (2) log2 transformation; and (3) the median of biological replicates. Extinction score <2 -3 After excluding the sgRNA and NAG PAM sgRNA, a QQ plot was generated using R software with a line fitted through the first and third quantiles. The sgRNA sequences were mapped to the human genome (hg19), and the cleavage position was set betw...

Claims

1. a. complementary to the plus or minus strand of positions 60725424-60725688 of human chromosome 2 (the +55 functional region); b. complementary to the plus or minus strand of positions 60722238 to 60722466 on human chromosome 2 (the +58 functional region); or c. A nucleic acid sequence complementary to the plus or minus strand of positions 60718042 to 60718186 of human chromosome 2 (the +62 functional region). A nucleic acid molecule comprising: the human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly; the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the genomic DNA sequence on human chromosome 2 at positions 60,716,189 to 60,728,612; Nucleic acid molecule.

2. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence excludes the entire BCL11A enhancer functional region.

3. 2. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence excludes the entirety of SEQ ID NOs: 136, 137 and 138.

4. 2. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence is short and is 13 base pairs (bp) or longer.

5. 2. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence is about 13 to 30 bp.

6. 2. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence is about 20 bp.

7. 2. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1-94.

8. 2. The nucleic acid molecule of claim 1, wherein the nucleic acid sequence further comprises a trans-activating CRISPR RNA (tracrRNA) sequence.

9. 2. The nucleic acid molecule of claim 1, which is a single guide RNA (sgRNA).

10. The nucleic acid molecule of claim 1, comprising a vector.

11. 11. The nucleic acid molecule of claim 10, wherein the vector is an sgRNA expression vector.

12. a. complementary to the plus or minus strand of positions 60725424-60725688 of human chromosome 2 (the +55 functional region); b. complementary to the plus or minus strand of positions 60722238 to 60722466 on human chromosome 2 (the +58 functional region); or c. A nucleic acid sequence complementary to the plus or minus strand of positions 60718042 to 60718186 of human chromosome 2 (the +62 functional region). A vector comprising: the human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly; the nucleic acid sequence excludes the entire human chromosome 2 and also excludes the genomic DNA sequence on human chromosome 2 at positions 60,716,189 to 60,728,612; vector.

13. The vector of claim 12, wherein the nucleic acid sequence excludes the entire BCL11A enhancer functional region.

14. 13. The vector of claim 12, wherein the nucleic acid sequence excludes the entirety of SEQ ID NOs: 136, 137 and 139.

15. 13. The vector of claim 12, wherein the nucleic acid sequence is short and is 13 base pairs (bp) or longer.

16. 13. The vector of claim 12, wherein the nucleic acid sequence is about 13 to 30 base pairs (bp).

17. 13. The vector of claim 12, wherein the nucleic acid sequence is about 20 bp.

18. 13. The vector of claim 12, wherein the nucleic acid sequence is selected from the group consisting of SEQ ID NOs: 1-94.

19. 13. The vector of claim 12, wherein the nucleic acid sequence further comprises a trans-activating CRISPR RNA (tracrRNA) sequence.

20. 13. The vector of claim 12, which is an sgRNA expression vector.

21. A method for producing progenitor cells having reduced BCL11A mRNA or protein expression, comprising contacting isolated progenitor cells with a nucleic acid molecule described in any one of claims 1 to 11 or a vector described in any one of claims 12 to 20.

22. 1. A method for producing progenitor cells with reduced BCL11A mRNA or BCL11A protein expression, comprising: contacting the isolated progenitor cells with an agent that binds to a human BCL11A enhancer functional region located on chromosome 2 at positions 60725424-60725688 (+55 functional region), 60722238-60722466 (+58 functional region), and / or 60718042-60718186 (+62 functional region); The agent is a. Positive or negative strand of human chromosome 2 at positions 60725424-60725688 (+55 functional region); b. the plus or minus strand of positions 60722238 to 60722466 of human chromosome 2 (the +58 functional region); or c. Positive or negative strand of human chromosome 2 at positions 60718042-60718186 (+62 functional region) binds to the human chromosome 2 is from the UCSC Genome Browser hg 19 human genome assembly; reducing BCL11A mRNA or protein expression by said step. A method comprising:

23. 23. The method of claim 21 or 22, further comprising contacting the isolated progenitor cells with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease.

24. 1. A method for producing an isolated genetically modified human cell having at least one genetic modification, comprising: contacting the isolated cells with an effective amount of a composition comprising the nucleic acid molecule of any one of claims 1 to 11 or the vector of any one of claims 12 to 20 together with at least one DNA targeting endonuclease or a vector carrying a coding sequence for a DNA targeting endonuclease, the DNA-targeting endonuclease cleaves the cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) to cause at least one genetic modification therein. Including, Human chromosome 2 from the UCSC Genome Browser hg 19 human genome assembly, method.

25. 25. The method of any one of claims 22 to 24, wherein the at least one DNA-targeting endonuclease is a Cas (CRISPR-associated) protein.

26. 26. The method of claim 25, wherein the Cas protein is Cas9.

27. The method of any one of claims 21 to 26, wherein the isolated progenitor cells or isolated cells are hematopoietic progenitor cells or hematopoietic stem cells.

28. 28. The method of claim 27, wherein the hematopoietic precursors are erythroid cells.

29. The method of any one of claims 21 to 26, wherein the isolated progenitor cells or isolated cells are induced pluripotent stem cells.

30. 30. The method of any one of claims 21 to 29, wherein the isolated progenitor cells or isolated cells are contacted ex vivo or in vitro.

31. 31. The method of any one of claims 21-30, wherein the contacted progenitor cell or the contacted cell acquires at least one genetic modification.

32. 30. The method of claim 29, wherein at least one genetic modification is a deletion, insertion or substitution of a nucleic acid sequence.

33. 33. The method of any one of claims 21-32, wherein the at least one genetic modification is located on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region).

34. The method of any one of claims 21 to 32, wherein the contacted progenitor cell or the contacted cell acquires at least one epigenetic modification in a BCL11A enhancer functional region.

35. 35. The method of claim 34, wherein the at least one epigenetic modification is selected from the group consisting of alterations in DNA methylation, histone tail modifications, histone subunit organization, and nucleosome positioning.

36. 36. The method of claim 34 or 35, wherein the at least one epigenetic modification is located on chromosome 2 at positions 60725424 to 60725688 (+55 functional region), at positions 60722238 to 60722466 (+58 functional region), and / or at positions 60718042 to 60718186 (+62 functional region).

37. 37. An isolated genetically modified human cell comprising at least one genetic modification on chromosome 2 at positions 60725424-60725688 (+55 functional region), at positions 60722238-60722466 (+58 functional region), and / or at positions 60718042-60718186 (+62 functional region) according to claims 21-36.

38. 38. A composition comprising the isolated genetically modified human cell of claim 37.

39. 1. A method for increasing fetal hemoglobin levels in a cell, comprising: contacting the isolated cells with an effective amount of a composition comprising the nucleic acid molecule of any one of claims 1 to 11 or the vector of any one of claims 12 to 20 together with at least one DNA targeting endonuclease or a vector carrying a coding sequence for a DNA targeting endonuclease, the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) to cause at least one genetic modification therein; said step increasing fetal hemoglobin expression in said cell or its progeny compared to said cell prior to said contacting; Including, Human chromosome 2 from the UCSC Genome Browser hg 19 human genome assembly, method.

40. 40. The method of claim 39, wherein the isolated cells are hematopoietic progenitor cells or hematopoietic stem cells.

41. 41. The method of claim 39 or 40, wherein the hematopoietic progenitor cells are erythroid cells.

42. 40. The method of claim 39, wherein the isolated cells are induced pluripotent stem cells.

43. 43. The method of any one of claims 39 to 42, wherein the isolated cells, hematopoietic progenitor cells, hematopoietic stem cells or induced pluripotent stem cells are contacted ex vivo or in vitro.

44. 44. The method of any one of claims 39-43, wherein the at least one DNA-targeting endonuclease is a Cas (CRISPR-associated) protein.

45. 45. The method of claim 44, wherein the Cas protein is Cas9.

46. 1. A method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising: contacting isolated hematopoietic progenitor cells in said mammal with an effective amount of a composition comprising the nucleic acid molecule of any one of claims 1 to 11 or the vector of any one of claims 12 to 20 together with at least one DNA-targeting endonuclease or a vector carrying a coding sequence for a DNA-targeting endonuclease, the DNA-targeting endonuclease cleaves cellular genomic DNA on chromosome 2 at positions 60725424-60725688 (+55 functional region), positions 60722238-60722466 (+58 functional region), and / or positions 60718042-60718186 (+62 functional region) to cause at least one genetic modification therein; said step increasing fetal hemoglobin expression in said mammal compared to the expression before said contacting; Including, Human chromosome 2 from the UCSC Genome Browser hg 19 human genome assembly, method.

47. 39. A method for increasing fetal hemoglobin levels in a mammal in need thereof, comprising transplanting into said mammal the isolated genetically modified human cell of claim 37 or the composition of claim 38.

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