COMPOSITIONS AND METHODS FOR TREATING SICKLE CELL DISEASE

1 . A method of treating a hemoglobinopathy in a patient in need thereof, the method comprising administering to the patient an effective amount of a pharmaceutically acceptable composition comprising a genetically modified cell, wherein the cell comprises a nucleic acid construct that inhibits the expression of NFIX within the cell.

2 . The method of claim 1 , wherein the hemoglobinopathy is a β-thalassemia.

3 . The method of claim 1 , wherein the hemoglobinopathy is sickle cell disease (SCD).

4 . The method of claim 1 , wherein the cell is a hematopoietic stem cell, a hematopoietic progenitor cell, or an erythroblast at any stage of development prior to enucleation.

5 . The method of claim 4 , wherein the cell is autologous to the patient.

6 . The method of claim 1 , wherein the nucleic acid construct comprises an shRNA or antisense nucleic acid sequence that inhibits the expression of NFIX

7 . The method of claim 6 , wherein the shRNA or antisense nucleic acid sequence base pairs with a sequence within the region of NFIX that encodes a DNA-binding domain or within the NFIX promoter/regulatory region (SEQ ID NO:761).

8 . The method of claim 7 , wherein the DNA-binding domain has the sequence

(SEQ ID [[NO:_]]NO: 1)

KQKWASRLLAKLRKDIRPEFREDFVLTITGKKPPCCVLSNPDQKGKIRR

IDCLRQADKVWRLDLVMVILFKGIPLESTDGERLYKSPQCSNPGLCVQP

HHIGV.

9 . The method of claim 6 , wherein the shRNA or antisense nucleic acid sequence base pairs with a sequence within Exon 2 and/or Exon 3 of NFIX.

10 . The method of claim 1 , wherein the nucleic acid construct comprises an shRNA comprising a sequence selected from the group consisting of:

(SEQ ID [[NO: ]]259)

CCGGGCAGTCTCAGTCCTGGTTCCTCTCGAGAGGAACCAGGACTGAGAC

TGCTTTTT;

(SEQ ID [[NO: ]]261)

CCGGACTGGATCTTTATCTGGCTTACTCGAGTAAGCCAGATAAAGATCC

AGTTTTTT;

(SEQ ID [[NO: ]]760)

CCGGCCGGCTTCTCTAAAGAAGTCACTCGAGTGACTTCTTTAGAGAAGC

CGGTTTTT;

(SEQ ID [[NO: ]]257)

CCGGCACATCACATTGGAGTCACAACTCGAGTTGTGACTCCAATGTGAT

GTGTTTTT;

(SEQ ID [[NO: ]]256)

CCGGCCTGTTGATGACGTGTTCTATCTCGAGATAGAACACGTCATCAAC

AGGTTTTT;

(SEQ ID [[NO: ]]262)

CCGGATCTTTATCTGGCTTACTTTGCTCGAGCAAAGTAAGCCAGATAAA

GATTTTTTG;

(SEQ ID [[NO:_]]2)

CCGGACATTGGAGTCACAATCAAAGCTCGAGCTTTGATTGTGACTCCAA

TGTTTTTTG;

(SEQ ID [[NO:_]]3)

CCGGGGAATCCGGACAATCAGATAGCTCGAGCTATCTGATTGTCCGGAT

TCCTTTTTG;

(SEQ ID [[NO:_]]263)

CCGGTAGGCTAAGAAACGCAGTATACTCGAGTATACTGCGTTTCTTAGC

CTATTTTTG;

and

(SEQ ID [[NO: ]]260)

CCGGCCCAACGGGCACTTAAGTTTCCTCGAGGAAACTTAAGTGCCCGTT

GGGTTTTTG.

or a sequence at least 90% identical thereto, optionally wherein the shRNA terminates at the residue immediately prior to the terminal poly-T sequence or comprises only the internally complementary sequences.

11 . The method of claim 10 , wherein the nucleic acid construct comprises an shRNA comprising

(SEQ ID [[NO:_]]2)

CCGGACATTGGAGTCACAATCAAAGCTCGAGCTTTGATTGTGACTCCAA

TGTTTTTTG

or

(SEQ ID [[NO:_]]3)

CCGGGGAATCCGGACAATCAGATAGCTCGAGCTATCTGATTGTCCGGAT

TCCTTTTTG.

12 . The method of claim 1 , wherein the genetically modified cell comprises the components of a nuclease-editing system.

13 . The method of claim 12 , wherein the nuclease-editing system is CRISPR or the genetically modified cell comprises a transposon.

14 . The method of claim 13 , wherein the genetically modified cell comprises a guide RNA that base pairs with a sequence within the region of NFIX that encodes a DNA-binding domain or within the NFIX promoter/regulatory region (SEQ ID NO:761).

15 . A non-naturally occurring, genetically modified cell, wherein the level of NFIX expression in the cell is decreased relative to that of a comparable, non-genetically modified cell of the same type.

16 . The cell of claim 15 , wherein the cell is a hematopoietic stem cell, a hematopoietic progenitor cell, or an erythroblast at any stage of development prior to enucleation.

17 . The cell of claim 15 , wherein the cell comprises an shRNA or antisense nucleic acid sequence that inhibits the expression of NFIX.

18 . A pharmaceutically acceptable composition comprising the cell of claim 15 .

19 . The pharmaceutically acceptable composition of claim 18 , formulated for intravenous administration.

20 . A kit comprising the cell of claim 15 , optionally within a pharmaceutically acceptable composition, and instructions for use.