Compositions and methods for immunooncology

Patent metadata

Field	Value
Publication number	US-12037583-B2
Application number	US-201615780751-A
Country	US
Kind code	B2
Filing date	Dec 2, 2016
Priority date	Dec 4, 2015
Publication date	Jul 16, 2024
Grant date	Jul 16, 2024

Abstract

The present invention is directed to genome editing systems, reagents and methods for immunooncology.

First claim

The invention claimed is:

1. A gRNA molecule comprising a tracr and a crRNA, wherein the crRNA comprises a targeting domain that is complementary to a target sequence of B2M, wherein the targeting domain comprises at least 19 consecutive nucleotides of a sequence selected from SEQ ID NO: 5498, SEQ ID NO: 5496, SEQ ID NO: 5499, or SEQ ID NO: 5509.

2. The gRNA molecule of claim 1 , wherein the targeting domain comprises SEQ ID NO: 5498, SEQ ID NO: 5496, SEQ ID NO: 5499, or SEQ ID NO: 5509.

3. The gRNA molecule of claim 1 , wherein the gRNA molecule comprises SEQ ID NO: 7858 or SEQ ID NO: 7853.

4. The gRNA molecule of claim 1 , wherein the targeting domain comprises SEQ ID NO: 5498.

5. The gRNA molecule of claim 1 , wherein the targeting domain and the tracr are disposed on separate nucleic acid molecules, and wherein the nucleic acid molecule comprising the targeting domain comprises SEQ ID NO: 6607, disposed immediately 3′ to the targeting domain, and wherein the nucleic acid molecule comprising the tracr comprises SEQ ID NO: 6660.

6. The gRNA molecule of claim 1 , wherein the gRNA molecule comprises one or more nucleic acid molecules comprising:
a) one or more phosphorothioate modification(s) at the 3′ end of said nucleic acid molecule or molecules;
b) one or more phosphorothioate modification(s) at the 5′ end of said nucleic acid molecule or molecules;
c) one or more 2′-O-methyl modification(s) at the 3′ end of said nucleic acid molecule or molecules;
d) one or more 2′-O-methyl modification(s) at the 5′ end of said nucleic acid molecule or molecules;
e) one or more 2′ O-methyl modification at each of the 4th-to-terminal, 3rd-to-terminal, and 2nd-to-terminal 3′ residues of said nucleic acid molecule or molecules; or
f) any combination thereof.

7. A nucleic acid comprising a sequence that encodes the gRNA molecule of claim 1 .

8. A vector comprising the nucleic acid of claim 7 .

9. The vector of claim 8 , wherein the vector is selected from the group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes simplex virus (HSV) vector, a plasmid, a minicircle, a nanoplasmid, and an RNA vector.

10. A composition comprising: (i) a gRNA molecule of claim 1 or nucleic acid comprising a sequence that encodes the gRNA molecule of claim 1 ; and (ii) a Cas9 molecule or a nucleic acid encoding a Cas9 molecule.

11. The composition of claim 10 , wherein the Cas9 molecule comprises any one of SEQ ID NO: 6611 or SEQ ID NO: 7821 to SEQ ID NO: 7831, or a sequence having at least 95% homology thereto.

12. The composition of claim 10 , wherein the gRNA molecule and Cas9 molecule are present in a ribonuclear protein complex (RNP).

13. The composition of claim 10 , further comprising a second gRNA molecule; a second gRNA molecule and a third gRNA molecule; or a second gRNA molecule, a third gRNA molecule, and a fourth gRNA molecule, wherein the second gRNA molecule, third gRNA molecule, and fourth gRNA molecule are each complementary to a different target sequence.

14. A composition comprising:
a) a first gRNA molecule comprising the gRNA molecule of claim 1 ; and
b) a second gRNA molecule comprising a tracr and a crRNA, wherein the crRNA of said second gRNA molecule comprises a targeting domain selected from any one of SEQ ID NO: 5528 to SEQ ID NO: 5623, SEQ ID NO: 5816 to SEQ ID NO: 5965, SEQ ID NO: 5624 to SEQ ID NO: 5643, SEQ ID NO: 5966 to SEQ ID NO: 6097, SEQ ID NO: 5644 to SEQ ID NO: 5719, SEQ ID NO: 6098 to SEQ ID NO: 6226, SEQ ID NO: 84 to SEQ ID NO: 392, SEQ ID NO: 393 to SEQ ID NO: 532, SEQ ID NO: 10780 to SEQ ID NO: 10794, SEQ ID NO: 533 to SEQ ID NO: 839, SEQ ID NO: 10677 to SEQ ID NO: 10764, SEQ ID NO: 840 to SEQ ID NO: 968, and SEQ ID NO: 10765 to SEQ ID NO: 10779.

15. The composition of claim 14 , wherein the targeting domain of said second gRNA molecule is selected from any one of SEQ ID NO: 5624 to SEQ ID NO: 5643 and SEQ ID NO: 5966 to SEQ ID NO:
6097.

16. The composition of claim 14 , further comprising a third gRNA molecule comprising a tracr and a crRNA, wherein the crRNA of said third gRNA molecule comprises a targeting domain selected from any one of SEQ ID NO: 6325 to SEQ ID NO: 6583, SEQ ID NO: 6662 to SEQ ID NO: 6749, SEQ ID NO: 6750 to SEQ ID NO: 7716, SEQ ID NO: 7717 to SEQ ID NO: 7804, and SEQ ID NO: 8622 to SEQ ID NO: 10089.

17. The composition of claim 16 , wherein the targeting domain of said third gRNA molecule is selected from any one of SEQ ID NO: 6750 to SEQ ID NO: 7716 and SEQ ID NO: 7717 to SEQ ID NO: 7804.

18. The composition of claim 16 , further comprising a fourth gRNA molecule comprising a tracr and a crRNA, wherein the crRNA of said fourth gRNA molecule comprises a targeting domain that is complementary to a target sequence of a target of an NK inhibitory molecule.

19. The composition of claim 18 , wherein the target of an NK inhibitory molecule is LILRB1.

20. The composition of claim 18 , wherein the targeting domain of said fourth gRNA molecule consists of:
a) any one of SEQ ID NO: 10090 to SEQ ID NO: 10673;
b) 17, 18, 19, 20, 21, 22, 23, or 24 consecutive nucleotides of any one of SEQ ID NO: 10090 to SEQ ID NO: 10673;
c) the 5′ 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, preferably 20 nucleotides, of any one of SEQ ID NO: 10090 to SEQ ID NO: 10673; or
d) the 3′ 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides, preferably 20 nucleotides, of any one of SEQ ID NO: 10090 to SEQ ID NO: 10673.

21. The composition of claim 10 , formulated in a medium suitable for electroporation.

22. The composition of claim 10 , further comprising a template nucleic acid.

23. The composition of claim 22 , wherein the template nucleic acid is in a vector.

24. The composition of claim 22 , wherein the template nucleic acid comprises a nucleic acid encoding a chimeric antigen receptor (CAR).

25. The composition of claim 24 , wherein the CAR is:
(a) a CD19 CAR;
(b) a BCMA CAR;
(c) a CD20 CAR;
(d) a CD22 CAR;
(e) a CD123 CAR;
(f) an EGFRvIII CAR; or
(g) a mesothelin CAR.

26. The composition of claim 22 , wherein the template nucleic acid comprises a nucleic acid encoding an NK inhibitory molecule.

27. A method of altering a target sequence of a cell, comprising contacting said cell with:
a) a gRNA molecule of claim 1 , and a Cas9 molecule;
b) a gRNA molecule of claim 1 , and a nucleic acid encoding a Cas9 molecule;
c) a nucleic acid encoding a gRNA molecule of claim 1 and a Cas9 molecule;
d) a nucleic acid encoding a gRNA molecule of claim 1 , and a nucleic acid encoding a Cas9 molecule;
e) any of a) to d), above, and a template nucleic acid; or
f) any of a) to d) above, and a nucleic acid comprising sequence encoding a template nucleic acid; and
(g) any one of a) to f), above, and one or more additional gRNA molecules.

28. The method of claim 27 , wherein the gRNA molecule or the nucleic acid encoding the gRNA molecule, and the Cas9 molecule or the nucleic acid encoding the Cas9 molecule, are formulated in more than one composition.

29. The method of claim 27 , wherein the gRNA molecule or the nucleic acid encoding the gRNA molecule, the Cas9 molecule or the nucleic acid encoding the Cas9 molecule, and, if present, the one or more additional gRNA molecule(s) and template nucleic acid are introduced into the cell by electroporation.

Assignees

• Novartis Ag
• Intellia Therapeutics Inc

Inventors

• Chen Ming-Wei
• Deck Melissa
• Dranoff Glenn
• Mickanin Craig Stephen
• Lescarbeau Reynald
• Richardson Celeste
• Stewart Morag Helen
• Yang Yi

Classifications

• A61K40/11

  T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells · CPC title

• A61K40/50

  characterised by the use of allogeneic cells · CPC title

• A61K40/4215

  Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30 · CPC title

• A61K40/4211

  CD19 or B4 · CPC title

• A61K40/421

  Immunoglobulin superfamily · CPC title