Methods and compositions for modulating gene expression

Patent metadata

Field	Value
Publication number	US-11759533-B2
Application number	US-201815940183-A
Country	US
Kind code	B2
Filing date	Mar 29, 2018
Priority date	Mar 29, 2017
Publication date	Sep 19, 2023
Grant date	Sep 19, 2023

Abstract

The present technology relates to compositions and methods for modulating expression of genes which include a target oligonucleotide sequence, such as repeats of a particular oligonucleotide sequence containing 3 to 10 nucleotides. In particular aspects, the present technology relates to agents having a formula A-L-B, wherein -L- is a linker; A- is a Brd4 binding moiety; and —B is a nucleic acid binding moiety, such as a polyamide or complementary oligonucleotide, that specifically binds to the target oligonucleotide sequence.

First claim

What is claimed is:

1. An agent having a formula A-L-B, wherein:
-L- is a linker;
A- is a Brd4 binding moiety selected from the group consisting of triazolodiazepines having a structure

wherein:
J is N, O or CR 11 ; K is N, O or CR 11 ; with the proviso that J and K cannot both be —O—; P is N, except when one of J or K is —O—, P is C, wherein R 11 is a hydrogen or optionally substituted alkyl, cycloalkyl, heterocycloalkyl, aryl, or heteroaryl group,
R 1 is a hydrogen or optionally substituted alkyl, hydroxyalkyl, aminoalkyl, alkoxyalkyl, halogenated alkyl, hydroxyl, alkoxy, or —COOR 4 , wherein R 4 is a hydrogen, substituted or unsubstituted arylene, aralkylene, cycloalkylene, heteroarylene, heteroaralkylene, heterocycloalkylene, alkylene, alkenylene, alkynylene, or cycloalkylalkylene group optionally interrupted by one or more heteroatoms,
R 2 is an optionally substituted aryl, alkyl, cycloalkyl, or aralkyl group,
R3 and R10 are each independently a hydrogen, halogen, or optionally substituted alkyl group,

“x” is an integer of from 1 to 10, and
Ring E is an optionally substituted aryl or heteroaryl ring; and
thienotriazolodiazepines having a structure

wherein:
R 2 is an aryl group optionally substituted with one or more substituted or unsubstituted alkyl, alkenyl, alkynyl, halogen, —OR 6 , —SR 6 , —N(R 6 ) 2 , —N(R 6 )COR 9 groups, wherein R 6 and R 9 are independently a hydrogen or alkyl group,
R 1 and R 3 are independently a hydrogen or optionally substituted alkyl group,
R 5 and R 7 are each independently a hydrogen, alkyl, alkenyl, alkynyl, halogen, halogenated alkyl, —OH, —SH, or —NH 2 , and
“x” is an integer of from 1 to 10; and
—B is a nucleic acid binding moiety that specifically binds to one or more repeats of a target oligonucleotide sequence selected from the group consisting of CGG, CTG, and CCTG.

2. The agent of claim 1 , wherein the nucleic acid binding moiety —B is a polyamide that specifically binds to the one or more repeats of the target oligonucleotide sequence.

3. The agent of claim 1 , wherein the nucleic acid binding moiety —B is a second oligonucleotide sequence that specifically binds to the target oligonucleotide sequence.

4. A pharmaceutical composition comprising a therapeutically effective amount of the agent of claim 1 and a pharmaceutically acceptable carrier.

5. A method for modulating mRNA levels produced from a gene in a cell comprising contacting the cell with an effective amount of the agent of claim 1 , wherein the gene comprises more than one repeat of the target oligonucleotide sequence.

6. The method of claim 5 , wherein the gene comprises at least about 30 repeats of the target oligonucleotide sequence.

7. The method of claim 5 , wherein the mRNA levels are increased by at least about 2.5-fold relative to an untreated cell.

8. A method for increasing levels of a protein in a cell, comprising contacting the cell with an effective amount of the agent of claim 1 , wherein the protein is encoded by a gene, wherein the gene comprises more than one repeat of the target oligonucleotide sequence.

9. The agent of claim 2 , wherein the nucleic acid binding moiety —B comprises a subunit selected from the group consisting of:

wherein Z is hydrogen, amino, or amido.

10. The agent of claim 9 , wherein Z is —H, —NR B R B , —N + R A R B R B , or —N(R A )C(O)R B ; wherein R A is hydrogen; and R B is selected from the group consisting of hydrogen, C 1 -C 6 alkyl, C 1 -C 6 alkenyl, and C 1 -C 6 alkynyl group.

11. The agent of claim 10 , wherein R B is hydrogen or —CH 3 .

12. The agent of claim 1 , wherein the linker -L- has a backbone chain comprising at least about 10 contiguous atoms.

13. The agent of claim 12 , wherein the linker -L- has a backbone chain comprising 15 to 30 contiguous atoms.

14. The agent of claim 13 , wherein the linker -L- comprises a combination of one or more optionally substituted arylene, aralkylene, cycloalkylene, heteroarylene, heteroaralkylene, heterocycloalkylene, alkylene, alkenylene, alkynylene, or cycloalkylalkylene groups, optionally interrupted by one or more heteroatoms, amido, or carboxyl groups.

15. The agent of claim 13 , wherein the linker -L- comprises a combination of one or more linking moieties selected from the group consisting of —O—, —(CH 2 ) x —, —(CH 2 CH 2 O) y —, —(OCH 2 CH 2 ) y —, —C(O)NR′—, —NR′C(O)—, —C(O)—, —NR*—,

—(CH 2 CH 2 CH 2 O) y —, and —(OCH 2 CH 2 CH 2 ) y —,
wherein R′ and R* are each independently a hydrogen or C 1 -C 6 alkyl; and
x and y are each independently an integer from 1-10.

16. The agent of claim 13 , wherein the linker -L- comprises
—(CH 2 ) x —C(O)N(R′)—(CH 2 ) Q —N(R*)—(CH 2 ) Q —N(R′)C(O)—(CH 2 ) x —C(O)N(R′)—,
—(CH 2 ) x —C(O)N(R′)—(CH 2 CH 2 O) y —(CH 2 ) x —C(O)N(R′)—,
—C(O)N(R′)—(CH 2 ) Q —N(R*)—(CH 2 ) Q —N(R′)C(O)—(CH 2 ) x —,
—(CH 2 ) x —O—(CH 2 CH 2 O) y —(CH 2 ) x —N(R′)C(O)—(CH 2 ) x —, or
—N(R′)C(O)—(CH 2 ) x —C(O)N(R′)—(CH 2 ) x —O—(CH 2 CH 2 O) y —(CH 2 ) x —;
wherein R* is methyl, R′ is hydrogen; y is an integer from 1 to 10; Q is an integer from 2 to 10; and x is an integer from 1 to 10.

17. The agent of claim 13 , wherein the linker -L- comprises one or more linking moieties selected from, Gly-Ser-Gly, (Gly-Ser-Gly)v (SEQ ID NOS:14 and 22-29), Gly-Gly-Ser, and (Gly-Gly-Ser)w (SEQ ID NOS:15 and 30-37), where v and w are independently integers from 2 to about 10.

18. The agent of claim 1 , wherein the Brd4 binding moiety A- has the structure:

19. The agent of claim 1 , wherein the Brd4 binding moiety A- is a thienotriazolodiazepine.

20. The agent of claim 1 , wherein the Brd4 binding moiety A- has the structure:

21. The agent of claim 1 , wherein R 2 is a phenyl group optionally substituted with one or more alkyl, halogenated alkyl, alkoxy, hydroxyalkyl, cyano, and/or halogen substituents.

22. The agent of claim 1 , wherein R 2 is a phenyl group optionally substituted with one or more halogens.

23. The agent of claim 1 , wherein the Brd4 binding moiety A- has the structure:

wherein R 3 is hydrogen or —CH 3 ;
R 1 , R 5 , and R 7 are each independently hydrogen, methyl, ethyl, or halomethyl group; and
R 8 is a halogen.

24. The agent of claim 23 , wherein R 3 is hydrogen or —CH 3 ; R 1 , R 5 , and R 7 are —CH 3 ; and R 8 is chloro.

25. The agent of claim 1 , wherein
L- comprises a backbone chain which includes at least about 10 contiguous atoms;
—B is a polyamide that specifically binds to one or more repeats of the target oligonucleotide sequence; and
A- is

Assignees

• Wisconsin Alumni Res Found

Inventors

• Ansari Aseem
• Erwin Graham
• Grieshop Matthew

Classifications

• C07D495/14 Primary

  Ortho-condensed systems · CPC title

• A61K48/0066 Primary

  Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61K47/60

  the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol · CPC title

• A61K47/545

  Heterocyclic compounds (A61K47/558 takes precedence) · CPC title