Targeting BCL11A distal regulatory elements for fetal hemoglobin reinduction
Provided herein are methods and compositions for increasing fetal hemoglobin levels in a cell by disrupting BCL11A expression at the genomic level. Also provided herein are methods and compositions relating to the treatment of hemoglobinopathies by reinduction of fetal hemoglobin levels.
1. A method for producing a genetically modified human hematopoietic progenitor cell having decreased BCL11A mRNA and protein expression, the method comprising introducing into an isolated human hematopoietic progenitor cell, in vitro or ex vivo, a vector comprising a regulatory element operably linked to the coding sequence of a DNA-targeting endonuclease, wherein the DNA-targeting endonuclease comprises a TAL DNA binding domain and whereby the DNA-targeting endonuclease binds to and cleaves the genomic DNA of the cell within chromosome 2 between positions 60,716,189-60,728,61 (according to UCSC Genome Browser hg 1.9 human genome assembly) thereby disrupting the DNAse hypersensitive sites +62, +58, or +55 therein, thereby genetically modifying the human hematopoietic progenitor cell and reducing the mRNA and the protein expression of BCL11A.
2. The method of claim 1 wherein the vector is introduced into the isolated human hematopoietic progenitor cell ex vivo.
3. A method for producing a genetically modified human hematopoietic progenitor cell having decreased BCL11A mRNA and protein expression, the method comprising introducing at least an RNA encoding a DNA-targeting endonuclease into an isolated hematopoietic progenitor cell in vitro or ex vivo, wherein the DNA-targeting, endonuclease comprises a TAL DNA binding domain, and whereby the DNA-targeting endonuclease binds to and cleaves the genomic DNA of the cell within chromosome 2 between positions 60,716,189-60,728,612 (according to UCSC Genome Browser hg 19 human genome assembly) thereby disrupting the DNAse hypersensitive sites +62, +58, or +55 therein, thereby genetically modifying the human hematopoietic progenitor cell and reducing the mRNA and the protein expression of BCL11A.
4. The method of claim 3 , wherein the DNA-targeting endonuclease is introduced into the isolated human hematopoietic progenitor cell ex vivo.
5. The method of claim 1 or claim 3 , wherein the isolated human hematopoietic progenitor is a cell of the erythroid lineage.
6. The method of claim 1 or claim 3 , wherein the isolated human hematopoietic progenitor cell comprises at least one genetic modification.
7. A method of increasing fetal hemoglobin levels in an isolated hematopoietic progenitor cell, the method comprising introducing into the isolated hematopoietic progenitor cell ex vivo a vector comprising a regulatory element operably linked to the coding sequence of a DNA-targeting endonuclease, wherein the DNA-targeting endonucleases comprises a TAL DNA binding domain and whereby the DNA-targeting endonuclease binds to and cleaves the genomic DNA of the hematopoietic progenitor cell within chromosome 2 between positions 60,716,189-60,728,61 causing at least one genetic modification in the DNAse hypersensitive sites +62, +58, or +55 therein, Whereby fetal hemoglobin expression is increased in the hematopoietic progenitor or erythroid progeny of the hematopoietic progenitor cell, compared to a hematopoietic progenitor cell or erythroid cell without the at least one genetic modification wherein the genetic modification is an insertion or a deletion.
8. A method of increasing fetal hemoglobin levels in an isolated hematopoietic progenitor cell, the method comprising introducing at least an RNA encoding a DNA-targeting endonuclease into an isolated hematopoietic progenitor cell ex vivo, wherein the DNA-targeting endonuclease comprises a TAL DNA binding domain, and whereby the DNA-targeting endonuclease binds and cleaves the genomic DNA of the hematopoietic progenitor cell within on chromosome 2 between positions 60,716,189-60,728,612 causing at least one genetic modification in the DNAse hypersensitive sites +62, +58, or +55 therein, whereby fetal hemoglobin expression is increased in the hematopoietic progenitor or erythroid progeny of the hematopoietic progenitor cell, compared to a hematopoietic progenitor cell or erythroid cell without the at least one genetic modification wherein the genetic modification is an insertion or a deletion.
9. The method of claim 6 , claim 7 or claim 8 , wherein the at least one genetic modification is a deletion.
10. The method of claim 9 , wherein the deletion removes the entire region between chromosome 2 location 60,716,189-60,728,612 or removes one or more DHSs in the region.
11. The method of claim 9 , wherein the deletion removes the entire region between chromosome 2 location 60,716,189-60,728,612.
12. The method of claim 9 , wherein the deletion disrupts DHS +62.
13. The method of claim 9 , wherein the deletion disrupts DHS +58.
14. The method of claim 9 , wherein the deletion disrupts DHS +55.