Materials and methods for treatment of hemoglobinopathies
The present application provides materials and methods for treating hemoglobinopathies. More specifically, the application provides methods for producing progenitor cells that are genetically modified via genome editing to increase the production of fetal hemoglobin (HbF), as well as modified progenitor cells (including, for example, CD34 + human hematopoietic stem cells) producing increased levels of HbF, and methods of using such cells for treating hemoglobinopathies such as sickle cell anemia and β-thalassemia.
1. A genetically modified human cell comprising:
(i) a deletion or insertion at one or more chromosomal sequences that are complementary to a nucleic acid sequence of any one of SEQ ID NOs: 161,203; 161,204; 161,205; 161,206; 161,207; 161,213; 161,215; 161,216; 161,217; 161,218; 161,220; 161,221; 161,222; 161,224; 161,230; 161,231; 161,309; 161,233; 161,235; 161,236; 161,237; 161,240; 161,241; 161,243; 161,245; 161,250; 161,252; 161,254; 161,255; 161,256; 161,257; 161,258; 161,259; 161,262; 161,265; 161,267; 161,269; 161,271; 161,272; 161,273; 161,274; 161,275; 161,278; 161,279; 161,280; 161,281; 161,283; 161,287; 161,297; 161,298; 161,299; 161,300; 161,303; 161,310; 161,311; and 161,312;
(ii) a Cas9 endonuclease and/or one or more guide RNAs (gRNAs);
wherein the cell is produced by a method comprising introducing the Cas9 endonuclease and the one or more gRNAs to the human cell to effect one or more double-strand breaks (DSBs) at one or more loci within the δβ-globin region of human chromosome 11, causing deletions or insertions of chromosomal DNA at the one or more loci, wherein one of the one or more gRNAs comprise a spacer sequence selected from the group consisting of a corresponding RNA sequence of any one of SEQ ID NOs: 161,203; 161,204; 161,205; 161,206; 161,207; 161,213; 161,215; 161,216; 161,217; 161,218; 161,220; 161,221; 161,222; 161,224; 161,230; 161,231; 161,309; 161,233; 161,235; 161,236; 161,237; 161,240; 161,241; 161,243; 161,245; 161,250; 161,252; 161,254; 161,255; 161,256; 161,257; 161,258; 161,259; 161,262; 161,265; 161,267; 161,269; 161,271; 161,272; 161,273; 161,274; 161,275; 161,278; 161,279; 161,280; 161,281; 161,283; 161,287; 161,297; 161,298; 161,299; 161,300; 161,303; 161,310; 161,311; and 161,312.
2. The modified human cell of claim 1 , wherein one of the one or more gRNAs comprise a spacer sequence selected from the group consisting of a corresponding RNA sequence of any one of SEQ ID NOs: 161,205; 161,206; 161,207; 161,217; 161,230; 161,231; 161,241; 161,254; 161,255; 161,259; 161,269; 161,275; 161,310; and 161,311.
3. The modified human cell of claim 1 , comprising the Cas9 endonuclease and the one or more gRNAs.
4. The modified human cell of claim 1 , wherein the modified human cell has increased expression of γ-globin and/or fetal hemoglobin (HbF) relative to an unmodified human cell.
5. The modified human cell of claim 1 , wherein the human cell is an isolated progenitor cell.
6. The modified human cell of claim 5 , wherein the isolated progenitor cell is a hematopoietic progenitor cell or an induced pluripotent stem cell.
7. The modified human cell of claim 1 , wherein the one or more loci are proximal to a boundary of the Corfu long deletion or the Corfu small deletion.
8. The modified human cell of claim 1 , wherein one of the loci consists of a sequence that is complementary to a nucleic acid sequence of any one of SEQ ID NOs: 161,203; 161,204; 161,205; 161,206; 161,207; 161,213; 161,215; 161,216; 161,217; 161,218; 161,220; 161,221; 161,222; 161,224; 161,230; 161,231; 161,309; 161,233; 161,235; 161,236; 161,237; 161,240; 161,241; 161,243; 161,245; 161,250; 161,252; 161,254; 161,255; 161,256; 161,257; 161,258; 161,259; 161,262; 161,265; 161,267; 161,269; 161,271; 161,272; 161,273; 161,274; 161,275; 161,278; 161,279; 161,280; 161,281; 161,283; 161,287; 161,297; 161,298; 161,299; 161,300; 161,303; 161,310; 161,311; and 161,312.
9. The modified human cell of claim 1 , wherein one of the loci comprises a sequence that is complementary to a nucleic acid sequence of any one of SEQ ID NOs: 161,205; 161,206; 161,207; 161,217; 161,230; 161,231; 161,241; 161,254; 161,255; 161,259; 161,269; 161,275; 161,310; and 161,311.
10. The modified human cell of claim 1 , wherein the cell is derived from a human patient having a β-hemoglobinopathy.
11. The modified human cell of claim 1 , wherein the cell is derived from a human patient having sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S/C disease, hemoglobin D disease, hemoglobin E disease, or a thalassemia.
12. A composition comprising a plurality of the modified human cell of claim 1 and a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable salt.