Compositions and methods for the treatment of hemoglobinopathies

Abstract

Provided are compositions and methods for the treatment of hemoglobinopathies such as thalassemias and sickle cell disease. Compositions and methods include one or more endonuclease(s) or endonuclease fusion protein(s), including one or more homing endonuclease(s) and/or homing endonuclease fusion protein(s) and/or CRISPR endonuclease(s) ad/or CRISPR endonuclease fusion protein(s): (a) to disrupt a Bcl11a coding region; (b) to disrupt a Bcl11a gene regulatory region; (c) to modify an adult human β-globin locus; (d) to disrupt a HbP silencing DNA regulatory element or pathway, such as a Bcl11a-regulated HbP silencing region; (e) to mutate one or more γ-globin gene promoter(s) to achieve increased expression of a γ-globin gene; (f) to mutate one or more δ-globin gene promoter(s) to achieve increased expression of a δ-globin gene; and/or (g) to correct one or more β-globin gene mutation(s).

1 - 96 . (canceled) 
     
     
         97 . A polynucleotide encoding an endonuclease selected from the group consisting of a homing endonuclease (HE) and a CRISPR endonuclease, wherein said endonuclease binds to a nucleotide sequence selected from the group consisting of a Bcl11a coding region, a Bcl11a gene regulatory region, an adult human β-globin locus, a fetal hemoglobin (HbF) silencing region, a Bcl11a-regulated HbF silencing region, a γ-globin gene promoter, a δ-globin gene promoter, and a site of a β-globin gene mutation. 
     
     
         98 . The polynucleotide of  claim 97 , wherein the endonuclease is:
 a) a HE that binds to said Bcl11a coding region or said Bcl11a gene regulatory region;   b) a HE selected from the group consisting of an I-OnuI homing endonuclease, an I-HjeMI homing endonuclease, and an I-CpaMI homing endonuclease;   c) a HE that can specifically bind to a fetal hemoglobin (HbF) silencing region;   d) an I-OnuI homing endonuclease;   e) an I-OnuI homing endonuclease comprising the amino acid sequence encoded by a variant of the nucleotide sequence of SEQ ID NO: 34 that encodes an I-OnuI homing endonuclease that can specifically bind to the fetal hemoglobin (HbF) silencing region;   f) an I-OnuI homing endonuclease comprising one or more amino acid substitutions within the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 34, wherein each of the amino acid substitutions is selected from the group consisting of L26, R28, R30, N32, S40, E42, G44, Q46, A70, S72, S78, K80, and T82; or   g) an I-OnuI homing endonuclease comprising one or more amino acid substitutions within the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 34, wherein said amino acid substitution is selected from the group consisting of F182, N184, 1186, S190, K191, Q197, V199, S201, K225, K227, D236, V238, and T240.   
     
     
         99 . The polynucleotide of  claim 97  or  claim 98 , further comprising a polynucleotide encoding a TAL effector nuclease (TALEN), a TALE-HE fusion protein, and/or a TREX2 nuclease. 
     
     
         100 . A vector system comprising a vector and:
 a) a polynucleotide encoding an endonuclease selected from the group consisting of a homing endonuclease (HE) and a CRISPR endonuclease, wherein said endonuclease binds to a nucleotide sequence selected from the group consisting of a Bcl11a coding region, a Bcl11a gene regulatory region, an adult human β-globin locus, a fetal hemoglobin (HbF) silencing region, a Bcl11a-regulated HbF silencing region, a γ-globin gene promoter, a δ-globin gene promoter, and a site of a β-globin gene mutation; or   b) a polynucleotide encoding a Cas9 endonuclease, optionally wherein the Cas9 endonuclease comprises the nucleotide sequence of SEQ ID NO: 37 or a variant thereof which encodes a functional Cas9 endonuclease, and an RNA guide strand that mediates the binding of the Cas9 endonuclease to a fetal hemoglobin (HbF) silencing region, optionally wherein the RNA guide strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54.   
     
     
         101 . The vector system of  claim 100 , wherein said endonuclease is:
 a) a HE that binds to said Bcl11a coding region or said Bcl11a gene regulatory region;   b) a HE selected from the group consisting of an I-OnuI homing endonuclease, an I-HjeMI homing endonuclease, and an I-CpaMI homing endonuclease;   c) a HE that can specifically bind to a fetal hemoglobin (HbF) silencing region;   d) an I-OnuI homing endonuclease;   e) an I-OnuI homing endonuclease comprising the amino acid sequence encoded by a variant of the nucleotide sequence of SEQ ID NO: 34 that encodes an I-OnuI homing endonuclease that can specifically bind to the fetal hemoglobin (HbF) silencing region;   f) an I-OnuI homing endonuclease comprising one or more amino acid substitutions within the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 34, wherein each of the amino acid substitutions is selected from the group consisting of L26, R28, R30, N32, S40, E42, G44, Q46, A70, S72, S78, K80, and T82; or   g) an I-OnuI homing endonuclease comprising one or more amino acid substitutions within the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 34, wherein said amino acid substitution is selected from the group consisting of F182, N184, 1186, S190, K191, Q197, V199, S201, K225, K227, D236, V238, and T240.   
     
     
         102 . The vector system of  claim 100 , further comprising a polynucleotide encoding a TAL effector nuclease (TALEN), a TALE-HE fusion protein, and/or a TREX2 nuclease. 
     
     
         103 . The vector system of  claim 100 ,  claim 101 , or  claim 102 , wherein said vector is selected from the group consisting of an AAV6, a modified adenovirus vector, an integration-deficient lentiviral vector (IDLV), and an integration-deficient foamyviral vector (IDFV). 
     
     
         104 . A polypeptide encoded by a polynucleotide encoding an endonuclease selected from the group consisting of a homing endonuclease (HE) and a CRISPR endonuclease, wherein said endonuclease binds to a nucleotide sequence selected from the group consisting of a Bcl11a coding region, a Bcl11a gene regulatory region, an adult human β-globin locus, a fetal hemoglobin (HbF) silencing region, a Bcl11a-regulated HbF silencing region, a γ-globin gene promoter, a δ-globin gene promoter, and a site of a β-globin gene mutation. 
     
     
         105 . The polypeptide of  claim 104 , wherein said endonuclease is:
 a) a HE that binds to said Bcl11a coding region or said Bcl11a gene regulatory region;   b) a HE selected from the group consisting of an I-OnuI homing endonuclease, an I-HjeMI homing endonuclease, and an I-CpaMI homing endonuclease;   c) a HE that can specifically bind to a fetal hemoglobin (HbF) silencing region;   d) an I-OnuI homing endonuclease;   e) an I-OnuI homing endonuclease comprising the amino acid sequence encoded by a variant of the nucleotide sequence of SEQ ID NO: 34 that encodes an I-OnuI homing endonuclease that can specifically bind to the fetal hemoglobin (HbF) silencing region;   f) an I-OnuI homing endonuclease comprising one or more amino acid substitutions within the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 34, wherein each of the amino acid substitutions is selected from the group consisting of L26, R28, R30, N32, S40, E42, G44, Q46, A70, S72, S78, K80, and T82; or   g) an I-OnuI homing endonuclease comprising one or more amino acid substitutions within the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 34, wherein said amino acid substitution is selected from the group consisting of F182, N184, 1186, S190, K191, Q197, V199, S201, K225, K227, D236, V238, and T240.   
     
     
         106 . A composition for the treatment of a hemoglobinopathy, said composition comprising:
 a) a polynucleotide encoding an endonuclease selected from the group consisting of a homing endonuclease (HE) and a CRISPR endonuclease, wherein said endonuclease binds to a nucleotide sequence selected from the group consisting of a Bcl11a coding region, a Bcl11a gene regulatory region, an adult human β-globin locus, a fetal hemoglobin (HbF) silencing region, a Bcl11a-regulated HbF silencing region, a γ-globin gene promoter, a δ-globin gene promoter, and a site of a β-globin gene mutation;   b) a vector system, comprising a vector and:
 i) a polynucleotide encoding an endonuclease selected from the group consisting of a homing endonuclease (HE) and a CRISPR endonuclease, wherein said endonuclease binds to a nucleotide sequence selected from the group consisting of a Bcl11a coding region, a Bcl11a gene regulatory region, an adult human β-globin locus, a fetal hemoglobin (HbF) silencing region, a Bcl11a-regulated HbF silencing region, a γ-globin gene promoter, a δ-globin gene promoter, and a site of a β-globin gene mutation; or 
 ii) a polynucleotide encoding a Cas9 endonuclease, optionally wherein the Cas9 endonuclease comprises the nucleotide sequence of SEQ ID NO: 37 or a variant thereof which encodes a functional Cas9 endonuclease, and an RNA guide strand that mediates the binding of the Cas9 endonuclease to a fetal hemoglobin (HbF) silencing region, optionally wherein the RNA guide strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54; or 
   c) a polypeptide encoded by a polynucleotide encoding an endonuclease selected from the group consisting of a homing endonuclease (HE) and a CRISPR endonuclease, wherein said endonuclease binds to a nucleotide sequence selected from the group consisting of a Bcl11a coding region, a Bcl11a gene regulatory region, an adult human β-globin locus, a fetal hemoglobin (HbF) silencing region, a Bcl11a-regulated HbF silencing region, a γ-globin gene promoter, a δ-globin gene promoter, and a site of a β-globin gene mutation.   
     
     
         107 . The composition of  claim 106  wherein said endonuclease is:
 a) a HE that binds to said Bcl11a coding region or said Bcl11a gene regulatory region; 
 b) a HE selected from the group consisting of an I-OnuI homing endonuclease, an I-HjeMI homing endonuclease, and an I-CpaMI homing endonuclease; 
 c) a HE that can specifically bind to a fetal hemoglobin (HbF) silencing region; 
 d) an I-OnuI homing endonuclease; 
 e) an I-OnuI homing endonuclease comprising the amino acid sequence encoded by a variant of the nucleotide sequence of SEQ ID NO: 34 that encodes an I-OnuI homing endonuclease that can specifically bind to the fetal hemoglobin (HbF) silencing region; 
 
     
     
         108 . The composition of  claim 106  wherein said polynucleotide further comprises a polynucleotide encoding a TAL effector nuclease (TALEN), a TALE-HE fusion protein, and/or a TREX2 nuclease. 
     
     
         109 . The composition of  claim 106  wherein said vector is selected from the group consisting of an AAV6, a modified adenovirus vector, an integration-deficient lentiviral vector (IDLV), and an integration-deficient foamyviral vector (IDFV). 
     
     
         110 . A cell comprising:
 a) a polynucleotide encoding an endonuclease selected from the group consisting of a homing endonuclease (HE) and a CRISPR endonuclease, wherein said endonuclease binds to a nucleotide sequence selected from the group consisting of a Bcl11a coding region, a Bcl11a gene regulatory region, an adult human β-globin locus, a fetal hemoglobin (HbF) silencing region, a Bcl11a-regulated HbF silencing region, a γ-globin gene promoter, a δ-globin gene promoter, and a site of a β-globin gene mutation;   b) a vector system comprising a vector and:
 i) a polynucleotide encoding an endonuclease selected from the group consisting of a homing endonuclease (HE) and a CRISPR endonuclease, wherein said endonuclease binds to a nucleotide sequence selected from the group consisting of a Bcl11a coding region, a Bcl11a gene regulatory region, an adult human β-globin locus, a fetal hemoglobin (HbF) silencing region, a Bcl11a-regulated HbF silencing region, a γ-globin gene promoter, a δ-globin gene promoter, and a site of a β-globin gene mutation; or 
 ii) a polynucleotide encoding a Cas9 endonuclease, optionally wherein the Cas9 endonuclease comprises the nucleotide sequence of SEQ ID NO: 37 or a variant thereof which encodes a functional Cas9 endonuclease, and an RNA guide strand that mediates the binding of the Cas9 endonuclease to a fetal hemoglobin (HbF) silencing region, optionally wherein the RNA guide strand comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, and SEQ ID NO: 54; or 
   c) a polypeptide encoded by a polynucleotide encoding an endonuclease selected from the group consisting of a homing endonuclease (HE) and a CRISPR endonuclease, wherein said endonuclease binds to a nucleotide sequence selected from the group consisting of a Bcl11a coding region, a Bcl11a gene regulatory region, an adult human β-globin locus, a fetal hemoglobin (HbF) silencing region, a Bcl11a-regulated HbF silencing region, a γ-globin gene promoter, a δ-globin gene promoter, and a site of a β-globin gene mutation.   
     
     
         111 . The cell of  claim 110  wherein said endonuclease is:
 a) a HE that binds to said Bcl11a coding region or said Bcl11a gene regulatory region; 
 b) a HE selected from the group consisting of an I-OnuI homing endonuclease, an I-HjeMI homing endonuclease, and an I-CpaMI homing endonuclease; 
 c) a HE that can specifically bind to a fetal hemoglobin (HbF) silencing region; 
 d) an I-OnuI homing endonuclease; 
 e) an I-OnuI homing endonuclease comprising the amino acid sequence encoded by a variant of the nucleotide sequence of SEQ ID NO: 34 that encodes an I-OnuI homing endonuclease that can specifically bind to the fetal hemoglobin (HbF) silencing region. 
 
     
     
         112 . The cell of  claim 110 , further comprising a polynucleotide encoding a TAL effector nuclease (TALEN), a TALE-HE fusion protein, and/or a TREX2 nuclease. 
     
     
         113 . The cell of  claim 110  wherein said vector is selected from the group consisting of an AAV6, a modified adenovirus vector, an integration-deficient lentiviral vector (IDLV), and an integration-deficient foamyviral vector (IDFV). 
     
     
         114 . The cell of  claim 110  wherein said cell is:
 a) a stem cell; 
 b) a stem cell selected from the group consisting of a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), an embryonic stem (ES) cell, and an erythroid progenitor cell; or 
 c) a stem cell selected from the group consisting of a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), and an erythroid progenitor cell. 
 
     
     
         115 . A genome edited stem cell wherein said genome edited stem cell is generated by the introduction of a homing endonuclease and a correction template. 
     
     
         116 . The genome edited stem cell of  claim 115  wherein said homing endonuclease is:
 a) a HE that binds to said Bcl11a coding region or said Bcl11a gene regulatory region; 
 b) a HE selected from the group consisting of an I-OnuI homing endonuclease, an I-HjeMI homing endonuclease, and an I-CpaMI homing endonuclease; 
 c) a HE that can specifically bind to a fetal hemoglobin (HbF) silencing region; 
 d) an I-OnuI homing endonuclease; 
 e) an I-OnuI homing endonuclease comprising the amino acid sequence encoded by a variant of the nucleotide sequence of SEQ ID NO: 34 that encodes an I-OnuI homing endonuclease that can specifically bind to the fetal hemoglobin (HbF) silencing region; 
 f) an I-OnuI homing endonuclease comprising one or more amino acid substitutions within the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 34, wherein each of the amino acid substitutions is selected from the group consisting of L26, R28, R30, N32, S40, E42, G44, Q46, A70, S72, S78, K80, and T82; or 
 g) an I-OnuI homing endonuclease comprising one or more amino acid substitutions within the amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 34, wherein said amino acid substitution is selected from the group consisting of F182, N184, 1186, S190, K191, Q197, V199, S201, K225, K227, D236, V238, and T240. 
 
     
     
         117 . The genome edited stem cell of  claim 115  wherein the homing endonuclease is fused to a TAL effector (TALE) DNA binding domain and/or a TREX2 nuclease domain. 
     
     
         118 . The genome edited stem cell of any one of  claims 115  to  117  wherein:
 a) the correction template comprises a nucleotide sequence that permits the modification of key regulatory or coding sequences within a globin gene locus; 
 b) the cell stem cell is selected from the group consisting of a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), an embryonic stem (ES) cell, and an erythroid progenitor cell; or 
 c) stem cell is selected from the group consisting of a hematopoietic stem cell (HSC), an induced pluripotent stem cell (iPSC), and an erythroid progenitor cell.