Compositions and methods for immunooncology

Patent metadata

Field	Value
Publication number	US-11851659-B2
Application number	US-201816498361-A
Country	US
Kind code	B2
Filing date	Mar 21, 2018
Priority date	Mar 22, 2017
Publication date	Dec 26, 2023
Grant date	Dec 26, 2023

Abstract

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

First claim

The invention claimed is:

1. A composition comprising:
a) a gRNA molecule or a nucleic acid sequence encoding the gRNA molecule, wherein the gRNA molecule comprises a tracr and crRNA, wherein the crRNA comprises a targeting domain that is complementary to a target sequence selected from a TET2 intron and a TET2 intron-exon junction, wherein the targeting domain is complementary to a sequence within a genomic region of chr4:105269748-105272563, wherein the location of the sequence within the genomic region is according to an alignment with the human reference genome hg38; and
b) a Cas9 molecule or a nucleic acid sequence encoding the Cas9 molecule, wherein the Cas9 molecule comprises an amino acid sequence of SEQ ID NO: 123.

2. The composition of claim 1 , wherein the targeting domain is complementary to a sequence within a genomic region selected from: chr4:105270624-105270643; chr4:105270630-105270649; chr4:105271863-105271883; chr4:105271340-105271360; chr4:105271204-105271223; chr4:105271526-105271546; chr4:105270350-105270370; chr4:105270268-105270288; chr4:105272182-105272202; chr4:105272465-105272485; chr4:105271387-105271407; chr4:105272436-105272456; chr4:105271924-105271944; chr4:105272323-105272343; chr4:105272057-105272077; chr4:105272309-105272329; chr4:105272324-105272344; chr4:105271184-105271204; chr4:105271190-105271210; chr4:105271295-105271315; chr4:105271292-105271312; chr4:105271458-105271478; chr4:105270635-105270655; chr4:105271173-105271192; chr4:105271232-105271252; chr4:105271845-105271865; chr4:105271849-105271869; and chr4:105271056-105271076.

3. The composition of claim 1 , wherein the targeting domain comprises or consists of any one of SEQ ID NO: 1000 to SEQ ID NO: 10514 or SEQ ID NO: 10515, or a fragment thereof.

4. The composition of claim 3 , wherein the fragment comprises 17, 18, 19, or 20 consecutive nucleic acids of any one of SEQ ID NO: 1000 to SEQ ID NO: 10514 or SEQ ID NO: 10515, wherein the 17, 18, 19, or 20 consecutive nucleic acids are disposed at the 3′ end or at the 5′ end of the targeting domain.

5. The composition of claim 1 , wherein a portion of the crRNA and a portion of the tracr hybridize to form a flagpole comprising SEQ ID NO: 50 or SEQ ID NO: 51.

6. The composition of claim 5 , wherein the flagpole further comprises a first flagpole extension, a second flagpole extension, or a combination thereof, located 3′ to the crRNA portion of the flagpole, wherein the first flagpole extension comprises SEQ ID NO: 55 and wherein the second flagpole extension comprises SEQ ID NO: 57, and wherein, if the gRNA comprises both the first flagpole extension and the second flagpole extension, the second flagpole extension is located 3′ to the first flagpole extension.

7. The composition of claim 1 , wherein the crRNA comprises, from 5′ to 3′, [targeting domain]-:
(a) SEQ ID NO: 50;
(b) SEQ ID NO: 51;
(c) SEQ ID NO: 77;
(d) SEQ ID NO: 78;
(e) SEQ ID NO: 79;
(f) SEQ ID NO: 80; or
(g) SEQ ID NO: 81; and/or
wherein the tracr comprises:
(h) SEQ ID NO: 53;
(i) SEQ ID NO: 54;
(j) SEQ ID NO: 82;
(k) SEQ ID NO: 83;
(l) SEQ ID NO: 65;
(m) SEQ ID NO: 84;
(n) SEQ ID NO: 87;
(o) SEQ ID NO: 76;
(p) SEQ ID NO: 85;
(q) SEQ ID NO: 86;
(r) any one of h) to q), above, further comprising, at the 3′ end, at least 1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides;
(s) any one of h) to q), above, further comprising, at the 3′ end, at least 1, 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides; or
(t) any one of h) to s), above, further comprising, at the 5′ end, at least 1, 2, 3, 4, 5, 6 or 7 adenine (A) nucleotides.

8. The composition of claim 7 , wherein the tracr comprises SEQ ID NO: 53 or SEQ ID NO: 54, and if a first flagpole extension is present, the gRNA further comprises a first tracr extension, disposed 5′ to SEQ ID NO: 53 or SEQ ID NO: 54, said first tracr extension comprising SEQ ID NO: 56.

9. The composition of claim 1 , wherein
(a) 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: 79 and the nucleic acid molecule comprising the tracr comprises SEQ ID NO: 65, or
(b) the targeting domain and the tracr are disposed on a single nucleic acid molecule, wherein the gRNA molecule comprises a loop disposed 3′ to the targeting domain and 5′ to the tracr, wherein the loop comprises SEQ ID NO: 52.

10. The composition of claim 1 , comprising, from 5′ to 3′, [targeting domain]-:
(a) SEQ ID NO: 71;
(b) SEQ ID NO: 72;
(c) SEQ ID NO: 73;
(d) SEQ ID NO: 74;
(e) SEQ ID NO: 75; or
(f) any of (a) to (e), above, further comprising, at the 3′ end, 1, 2, 3, 4, 5, 6 or 7 uracil (U) nucleotides.

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

12. The composition of claim 11 , wherein the gRNA molecule comprises a combination of any of a-e.

13. The composition of claim 1 , formulated in a medium suitable for electroporation.

14. The composition of claim 1 , wherein the gRNA molecule and the Cas9 molecule are present in a ribonuclear protein complex (RNP).

15. The composition of claim 1 , further comprising one or more additional gRNA molecules or one or more additional nucleic acid molecules encoding the one or more additional gRNA molecules.

16. The composition of claim 15 , wherein the one or more additional gRNA molecules comprises a targeting domain complementary to one or more of: a target sequence of an inhibitory molecule, PDCD1, a component of the T cell receptor, TRAC, TRBC, B2M, HLA-DM, HLA-DO, HLA-DR, HLA-DQ, HLA-DP, CIITA, RFXANK, RFXAP, RFX1, RFX5, NF-YA, NF-YB, NF-YC, X2BP, OCAB, HLA-A, HLA-B, HLA-C, NLRC5, CD247, CD3, CD3D, CD3E, CD3G, DCK, CD52, FKBP1A, and NR3C1.

17. The composition of claim 15 , wherein each gRNA molecule is in an RNP complex with a Cas9 molecule, and wherein each RNP complex is at a concentration of 10 μM or less.

18. The composition of claim 1 , wherein the composition further comprises a template nucleic acid, wherein the template nucleic acid is present in a vector and wherein the vector is a lentivirus vector, an AAV vector, an AAV6 vector, an adenovirus vector, a plasmid, a minicircle or a nanoplasmid.

19. The composition of claim 18 , wherein the template nucleic acid comprises at least one 5′ homology arm, at least one 3′ homology arm, or a combination thereof, wherein said homology arm comprises sequence homologous to sequence of a TET2 intron.

20. The composition of claim 18 , wherein:
(a) the template nucleic acid sequence is in an AAV6 vector;
(b) the template nucleic acid sequence comprises a nucleic acid sequence encoding a CAR selected from a CD19 CAR, a BCMA CAR, and a CD22 CAR; and/or
(c) the template nucleic acid sequence comprises a first homology arm comprising SEQ ID NO: 124 and a second homology arm comprising S

Assignees

• Novartis Ag
• Intellia Therapeutics Inc

Inventors

• Brogdon Jennifer
• Chen Ming-Wei
• Lim Hyungwook
• Yang Yi
• Stewart Morag
• Hesse Sarah

Classifications

• A61K40/30

  characterised by the recombinant expression of specific molecules in the cells of the immune system · CPC title

• A61K40/4215

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

• A61K40/31

  Chimeric antigen receptors [CAR] · CPC title

• A61K40/11

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

• A61K2300/00

  Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00 · CPC title