Compositions and methods to treating hemoglobinopathies
Embodiments herein provide specially designed synthetic BCL11A-targeting microRNAs for RNA polymerase II expression, and methods of use to treat hemoglobinopathies such as sickle cell disease or thalassemia by increasing the expression levels of fetal hemoglobin levels.
1. A method of treating, or reducing a risk of developing, a hemoglobinopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one synthetic BCL11A microRNA comprising:
a) a first BCL11A segment, a loop segment; and
b) a second BCL11A segment arranged in tandem in a 5′ to 3′ direction,
wherein the loop segment is between and directly linked to the first and second BCL11A segments,
wherein the first BCL11A segment is complementary to the second BCL11A segment so that the first and second BCL11A segments base pair to form a hairpin loop with the loop segment forming the loop portion of the hairpin loop thus formed; and
wherein the first BCL11A segment starts with a -GCGC- at the 5′ end and the second BCL11A segment ends with a -GCGC- at the 3′ end;
thereby treating, or reducing the risk of developing, the hemoglobinopathy in the subject.
2. The method of claim 1 , wherein the first and second BCL11A segments are about 18 to 25 nucleotides long.
3. The method of claim 1 , wherein the first BCL11A segment contains a sequence derived from a BCL11A mRNA sequence.
4. The method of claim 1 , wherein the first BCL11A segment comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 57, 58, 59, 60, 61, 62, 63, 64, and 65.
5. The method of claim 1 , wherein the first BCL11A segment comprises the nucleotide sequence set forth in SEQ ID NO: 61.
6. The method of claim 1 , wherein the loop segment is derived from a microRNA.
7. The method of claim 1 , wherein the loop segment is derived from a microRNA selected from the group consisting of: miR-142, miR-155, miR-181 and miR-223.
8. The method of claim 1 , wherein the loop segment is derived from miR223.
9. The method of claim 1 , wherein the loop segment comprises the polynucleotide sequence set forth in SEQ ID NO: 68.
10. The method of claim 1 , wherein the microRNA comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 41, and 42.
11. The method of claim 1 , wherein the microRNA comprises the polynucleotide sequence set forth SEQ ID NO: 39.
12. A method of treating, or reducing a risk of developing, a hemoglobinopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of a hematopoietic stem or progenitor cell comprising a lentiviral vector that comprises a nucleic acid encoding:
a) a first BCL11A segment, a loop segment; and
b) a second BCL11A segment arranged in tandem in a 5′ to 3′ direction,
wherein the loop segment is between and directly linked to the first and second BCL11A segments,
wherein the first BCL11A segment is complementary to the second BCL11A segment so that the first and second BCL11A segments base pair to form a hairpin loop with the loop segment forming the loop portion of the hairpin loop thus formed; and
wherein the first BCL11A segment starts with a -GCGC- at the 5′ end and the second BCL11A segment ends with a -GCGC- at the 3′ end;
thereby treating, or reducing the risk of developing, the hemoglobinopathy in the subject.
13. The method of claim 12 , wherein the cell is an erythroid cell.
14. The method of claim 12 , wherein the first and second BCL11A segments are about 18 to 25 nucleotides long.
15. The method of claim 12 , wherein the first BCL11A segment contains a sequence derived from a BCL11A mRNA sequence.
16. The method of claim 12 , wherein the first BCL11A segment comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 57, 58, 59, 60, 61, 62, 63, 64, and 65.
17. The method of claim 12 , wherein the first BCL11A segment comprises the nucleotide sequence set forth in SEQ ID NO: 61.
18. The method of claim 12 , wherein the loop segment is derived from a microRNA.
19. The method of claim 12 , wherein the loop segment is derived from a microRNA selected from the group consisting of: miR-142, miR-155, miR-181 and miR-223.
20. The method of claim 12 , wherein the loop segment is derived from miR223.
21. The method of claim 12 , wherein the loop segment comprises the polynucleotide sequence set forth in SEQ ID NO: 68.
22. The method of claim 12 , wherein the nucleic acid comprises a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 41, and 42.
23. The method of claim 12 , wherein the nucleic acid comprises the polynucleotide sequence set forth SEQ ID NO: 39.
24. A method of increasing fetal hemoglobin levels in a subject, the method comprising administering to the subject a therapeutically effective amount of a hematopoietic stem or progenitor cell comprising a lentiviral vector that comprises a nucleic acid encoding:
a) a first BCL11A segment, a loop segment; and
b) a second BCL11A segment arranged in tandem in a 5′ to 3′ direction,
wherein the loop segment is between and directly linked to the first and second BCL11A segments,
wherein the first BCL11A segment is complementary to the second BCL11A segment so that the first and second BCL11A segments base pair to form a hairpin loop with the loop segment forming the loop portion of the hairpin loop thus formed; and
wherein the first BCL11A segment starts with a -GCGC- at the 5′ end and the second BCL11A segment ends with a -GCGC- at the 3′ end;
thereby increasing fetal hemoglobin levels in the subject.
25. The method of claim 24 , wherein the nucleic acid comprises the polynucleotide sequence set forth SEQ ID NO: 39.
26. A method of increasing fetal hemoglobin levels in a subject, the method comprising administering to the subject a therapeutically effective amount of at least one synthetic BCL11A microRNA comprising:
a) a first BCL11A segment, a loop segment; and
b) a second BCL11A segment arranged in tandem in a 5′ to 3′ direction,
wherein the loop segment is between and directly linked to the first and second BCL11A segments,
wherein the first BCL11A segment is complementary to the second BCL11A segment so that the first and second BCL11A segments base pair to form a hairpin loop with the loop segment forming the loop portion of the hairpin loop thus formed; and
wherein the first BCL11A segment starts with a -GCGC- at the 5′ end and the second BCL11A segment ends with a -GCGC- at the 3′ end;
thereby increasing fetal hemoglobin levels in the subject.
27. The method of claim 26 , wherein the microRNA comprises the polynucleotide sequence set forth SEQ ID NO: 39.