Granted Patent
B2
US 12,241,087 · App. 17/898,540 · Granted Mar 4, 2025
Compositions and methods for differentiating stem cells into NK cells
Inventors:
Viktoriia Kyrychenko (Cambridge, MA); Wai Lun Leung (Cambridge, MA); Patrick Claudio Ovando Roche (Cambridge, MA)
Assignee:
CRISPR THERAPEUTICS AG
C12N5/0646A61K39/4613A61K39/4644C12N5/0647C12N2500/22C12N2500/34C12N2501/115C12N2501/125C12N2501/145C12N2501/155C12N2501/16C12N2501/165C12N2501/2303C12N2501/2307C12N2501/2315C12N2501/26C12N2501/415C12N2501/727C12N2506/02C12N2506/03C12N2506/45C12N2527/00
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Abstract
The disclosure features methods and compositions for differentiating stem cells into hematopoietic stem and progenitor cells (HSPC) and/or Natural Killer (NK) cells. The methods and compositions described herein are used to differentiate stem or progenitor cells having at least one gene-edit that is maintained in the differentiated cell. Also provided are differentiated cells produced using the methods and compositions described herein for therapeutic applications.
Claims (28)
1. A method for generating Natural Killer (NK) cells from pluripotent stem cells, the method comprising:
(a) culturing a population of pluripotent stem cells in a first medium comprising a ROCK inhibitor under conditions sufficient to form aggregates;
(b) culturing the aggregates in a second medium comprising BMP-4;
(c) culturing the aggregates in a third medium comprising BMP-4, FGF2, a WNT pathway activator, and Activin A;
(d) culturing the aggregates in a fourth medium comprising FGF2, VEGF, TPO, SCF, IL-3, FLT3L, and an activin/nodal inhibitor to form a cell population comprising hematopoietic stem and progenitor cells (HSPCs);
(e) culturing the cell population in a fifth medium comprising FGF2, VEGF, TPO, SCF, IL-3 and FLT3L;
(f) culturing the cell population in a sixth medium comprising IL-3, IL-7, FLT3L, IL-15 and SCF; and
(g) culturing the cell population in a seventh medium comprising IL-7, FLT3L, IL-15 and SCF; thereby generating NK cells.
2. The method of claim 1 , wherein culturing the cell population in the sixth medium in step (f) results in the formation of a progenitor cell population comprising common lymphoid progenitor (CLP) cells.
3. The method of claim 1 , wherein the ROCK inhibitor is thiazovivin or Y27632, the WNT pathway activator is CHIR-99021, and/or the activin/nodal inhibitor is SB-431542.
4. The method of claim 1 , wherein the second medium further comprises a ROCK inhibitor.
5. The method of claim 1 , wherein (a) comprises culturing for 12-48 hours; (b) comprises culturing for up to 24 hours; (c) comprises culturing for 1-3 days; (d) comprises culturing for 1-3 days; (e) comprises culturing for 1-3 days; (f) comprises culturing for at least 6 days and up to 8 days; or (g) comprises culturing for up to 6 days; or any combination of (a), (b), (c), (d), (e), (f), or (g).
6. The method of claim 1 , wherein the method is carried out under suspension agitation.
7. The method of claim 1 , wherein the sixth and/or seventh medium comprises human serum, zinc sulfate, ethanolamine, glucose, or any combination thereof; and/or the sixth and/or seventh medium comprises DMEM (high glucose)/F12 medium, and a supplement of human serum, zinc sulfate, ethanolamine, glucose or any combination thereof.
8. The method of claim 1 , wherein the fourth medium comprises 20 ng/mL FGF, 20 ng/mL VEGF, 20 ng/mL TPO, 100 ng/mL SCF, 40 ng/mL IL-3, 10-20 ng/mL FLT3L, and 5 μM SB-431542.
9. The method of claim 1 , wherein the fifth medium comprises 20 ng/mL FGF, 20 ng/mL VEGF, 20 ng/mL TPO, 100 ng/mL SCF, 40 ng/ml IL-3, and 10-20 ng/mL FLT3L.
10. The method of claim 1 , wherein the sixth medium comprises 20 ng/mL IL-7, 10-20 ng/mL FLT3L, 10-20 ng/ml IL-15, 20 ng/mL SCF, and 5 ng/ml IL-3.
11. The method of claim 1 , wherein the seventh medium comprises 20 ng/ml IL-7, 10-20 ng/mL FLT3L, 10-20 ng/ml IL-15, and 20 ng/mL SCF.
12. The method of claim 1 , comprising culturing the cell population in an eighth medium comprising IL-7, FLT3L, IL-15 and SCF for at least 6 days and up to 10-16 days total.
13. The method of claim 1 , wherein the NK cells express (a) at least one of: CD56 and CD45, and/or (b) at least one of: an activating receptor, an inhibitory receptor and a co-receptor.
14. The method of claim 13 , wherein the activating receptor is selected from the group of NKp44, NKp46, NKG2D, CD16, KIR2DL4, NKp30, and any combination thereof; the inhibitory receptor is selected from the group of NKG2A, KIR3DL2, and any combination thereof; and/or the co-receptor is CD94.
15. The method of claim 1 , wherein the NK cells have a function of (a) the ability to induce cell lysis and cell death of a target cell; (b) degranulation; or (c) a combination thereof.
16. The method of claim 1 , wherein the NK cells are generated without sorting CD34 + cells from the cell population.
17. The method of claim 1 , wherein the population of pluripotent stem cells comprises genetically modified stem cells.
18. The method of claim 1 , wherein the population of pluripotent stem cells comprises induced pluripotent stem cells (iPSC), embryonic stem cells (ESC), and/or adult stem cells (ASC).
19. The method of claim 1 , wherein the pluripotent stem cells are mammalian cells.
20. A plurality of Natural Killer (NK) cells generated by the method of claim 1 .
21. A method comprising administering to a subject the plurality of NK cells of claim 20 .
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Dec 12, 2024
From: KYRYCHENKO, VIKTORIIA; LEUNG, WAI LUN; OVANDO ROCHE, PATRICK CLAUDIO
To: CRISPR THERAPEUTICS AG
Reel/Frame 069573/0069 →
Continuity (4)
Provisional Application
63250037
· Sep 29, 2021
Provisional Application
63132230
· Dec 30, 2020
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