Targeting bcl11a enhancer functional regions for fetal hemoglobin reinduction

Abstract

Provided herein are nucleic acid molecules that target the BCL11A enhancer functional regions, compositions comprising the nucleic acid molecules and methods 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. In particular, the nucleic acid molecules target the +62, +58, and/or the +55 enhancer functional regions.

1 .- 47 . (canceled) 
     
     
         48 . A method for producing an isolated genetic engineered human cell having decreased BCL11A mRNA or protein expression, the method comprising expressing in a cell:
 a. a vector for gene editing comprising i) a protospacer adjacent motif (PAM) sequence; ii) a trans-activating CRISPR RNA (tracrRNA) sequence; and ii) a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1-94, and   b. at least a DNA-targeting endonuclease in an isolated human cell,   
       whereby the DNA-targeting endonuclease cleaves the genomic DNA of the cell on chromosome 2 at location 60725424 to 60725688 (+55 functional region), at location 60722238 to 60722466 (+58 functional region), and/or at location 60718042 to 60718186 (+62 functional region), causing at least one genetic modification therein, wherein the human chromosome 2 is that according to UCSC Genome Browser hg 19 human genome assembly. 
     
     
         49 . The method of  claim 48 , wherein the at least a DNA-targeting endonuclease is a CRISPR-associated (Cas) protein. 
     
     
         50 . The method of  claim 49 , the Cas protein is Cas9. 
     
     
         51 . The method of  claim 48 , wherein the vector is a virus. 
     
     
         52 . The method of  claim 48 , wherein the viral vector is a lentivirus, a retrovirus, an adenovirus, or adeno-associated virus. 
     
     
         53 . The method of  claim 48 , wherein the isolated cell is an embryonic stem cell, a somatic stem cell, a progenitor cell, a bone marrow cell, a hematopoietic progenitor cell or a hematopoietic stem cell. 
     
     
         54 . The method of  claim 48 , wherein the at least one genetic modification is a deletion, insertion or substitution of the nucleic acid sequence. 
     
     
         55 . The method of  claim 48 , wherein the cell acquires at least one epigenetic modification in the BCL11A enhancer functional region upon expression of the vector for gene editing and at least a DNA-targeting endonuclease in the cell. 
     
     
         56 . An isolated genetic engineered human cell having at least one genetic modification on chromosome 2 location 60725424 to 60725688 (+55 functional region), at location 60722238 to 60722466 (+58 functional region), and/or at location 60718042 to 60718186 (+62 functional region) according to  claim 48 . 
     
     
         57 . A composition comprising isolated genetic engineered human cells of  claim 56 . 
     
     
         58 . A method of increasing fetal hemoglobin levels in a cell, the method comprising decreasing the BCL11A mRNA or BCL11A expression in an isolated cell according to the method  claim 48 . 
     
     
         59 . The method of  claim 58 , wherein the isolated cell is an embryonic stem cell, a somatic stem cell, a progenitor cell, a bone marrow cell, a hematopoietic progenitor cell or hematopoietic stem cell. 
     
     
         60 . A method for increasing fetal hemoglobin levels in a mammal in need thereof, the method comprising transplanting a composition of  claim 57  into the mammal.