Compositions and methods for the treatment of hemoglobinopathies

Patent metadata

Field	Value
Publication number	US-12559748-B2
Application number	US-202217808802-A
Country	US
Kind code	B2
Filing date	Jun 24, 2022
Priority date	Feb 6, 2017
Publication date	Feb 24, 2026
Grant date	Feb 24, 2026

Abstract

The present invention is directed to genome editing systems, reagents and methods for the treatment of hemoglobinopathies.

First claim

The invention claimed is:

1 . A method of altering a cell at or near a target sequence within said cell, comprising contacting said cell with:
1) a guide RNA (gRNA) molecule comprising a tracr and crRNA, wherein the crRNA comprises a targeting domain that comprises SEQ ID NO: 67 and a Cas9 molecule; 2) a gRNA molecule comprising a tracr and crRNA, wherein the crRNA comprises a targeting domain that comprises SEQ ID NO: 67 and a polynucleotide comprising a nucleic acid sequence encoding a Cas9 molecule; 3) one or more polynucleotides comprising one or more nucleic acid sequences encoding a gRNA molecule comprising a tracr and crRNA, wherein the crRNA comprises a targeting domain that comprises SEQ ID NO: 67 and a Cas9 molecule; 4) one or more polynucleotides comprising one or more nucleic acid sequences encoding a gRNA molecule comprising a tracr and crRNA, wherein the crRNA comprises a targeting domain that comprises SEQ ID NO: 67 and a polynucleotide comprising a nucleic acid sequence encoding a Cas9 molecule; 5) any of 1) to 4), above, and a template nucleic acid; or 6) any of 1) to 4) above, and a polynucleotide comprising a nucleic acid sequence encoding a template nucleic acid.

2 . The method of claim 1 , wherein:
(a) the method results in a population of cells wherein at least about 15% of the cells of the population comprise an indel wherein the cells of the population do not comprise a deletion of a nucleotide disposed between 5,250,092 and 5,249,833, − strand (hg38); (b) the altering results in a cell that is capable of differentiating into a differentiated cell of an erythroid lineage, and wherein said differentiated cell exhibits an increased level of fetal hemoglobin relative to an unaltered cell; (c) the altering results in a population of cells that is capable of differentiating into a population of differentiated cells, and wherein said population of differentiated cells has an increased percentage of F cells relative to a population of unaltered cells; and/or (d) the altering results in a cell that is capable of differentiating into a differentiated cell and wherein said differentiated cell produces at least about 6 picograms fetal hemoglobin per cell.

3 . A method of preparing a cell comprising:
(a) providing a cell; (b) culturing said cell ex vivo in a cell culture medium comprising a stem cell expander; and (c) introducing into said cell a gRNA molecule comprising a tracr and crRNA, wherein the crRNA comprises a targeting domain that comprises SEQ ID NO: 67.

4 . The method of claim 1 , wherein the Cas9 molecule comprises the sequence of:
(a) SEQ ID NO: 233; (b) SEQ ID NO: 234; (c) SEQ ID NO: 235; (d) SEQ ID NO: 236; (e) SEQ ID NO: 237; (f) SEQ ID NO: 238; (g) SEQ ID NO: 239; (h) SEQ ID NO: 240; (i) SEQ ID NO: 241; (j) SEQ ID NO: 242; (k) SEQ ID NO: 243; or (l) SEQ ID NO: 244.

5 . The method of claim 1 , wherein the cell is a human cell.

6 . The method of claim 5 , wherein the cell is obtained from a patient suffering from a hemoglobinopathy.

7 . The method of claim 1 , wherein the cell is an HSPC.

8 . The method of claim 7 , wherein the cell is a CD34+ HSPC.

9 . The method of claim 8 , wherein the cell is a CD34+CD90+ HSPC.

10 . The method of claim 1 , wherein the cell has been isolated from bone marrow, peripheral blood, or umbilical cord blood.

11 . The method of claim 1 , wherein the cell is autologous or allogenic with respect to a patient to be administered said cell.

12 . The method of claim 1 , wherein the template nucleic acid is a nucleic acid encoding:
(a) human beta globin or a human beta globin including one or more of the mutations G16D, E22A and T87Q, or a fragment thereof, or (b) human gamma globin, or a fragment thereof.

13 . The method of claim 3 , wherein the stem cell expander is:
a) (1r,40-N1-(2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl)cyclohexane-1,4-diamine; b) methyl 4-(3-piperidin-1-ylpropylamino)-9H-pyrimido[4,5-b]indole-7-carboxylate; c) 4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol; d) (S)-2-(6-(2-(1H-indol-3-yl)ethylamino)-2-(5-fluoropyridin-3-yl)-9H-purin-9-yl)propan-1-ol; or e) a combination thereof.

14 . The method of claim 3 , wherein the cell culture medium comprises a stem cell expander at a concentration ranging from about 1 nM to about 1 mM.

15 . The method of claim 3 , wherein the cell provided in step (a) is a human cell.

16 . The method of claim 15 , wherein the cell is obtained from a patient suffering from a hemoglobinopathy.

17 . The method of claim 3 , wherein the cell is an HSPC.

18 . The method of claim 17 , wherein the cell is a CD34+ HSPC.

19 . The method of claim 18 , wherein the cell is a CD34+CD90+ HSPC.

20 . The method of claim 1 , wherein the gRNA molecule comprises SEQ ID NO: 195 disposed 3′ to the targeting domain.

21 . The method of claim 1 , wherein the gRNA molecule comprises SEQ ID NO: 231 disposed 3′ to the targeting domain.

22 . The method of claim 1 , wherein the gRNA molecule comprises:
(a) SEQ ID NO: 174; (b) SEQ ID NO: 175; or (c) SEQ ID NO: b 176 .

23 . The method of claim 3 , wherein the gRNA molecule comprises SEQ ID NO: 195 disposed 3′ to the targeting domain.

24 . The method of claim 3 , wherein the gRNA molecule comprises SEQ ID NO: 231 disposed 3′ to the targeting domain.

25 . The method of claim 3 , wherein the gRNA molecule comprises:
(a) SEQ ID NO: 174; (b) SEQ ID NO: 175; or (c) SEQ ID NO: 176.

Assignees

• Novartis Ag
• Intellia Therapeutics Inc

Inventors

• Mickanin Craig Stephen
• Schmedt Christian
• Snead Jennifer
• Stevenson Susan C
• Yang Yi

Classifications

• C12N2320/11

  for the determination of target sites, i.e. of active nucleic acids · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• A61K31/713

  Double-stranded nucleic acids or oligonucleotides · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title