Tunable endogenous protein degradation

Patent metadata

Field	Value
Publication number	US-11293023-B2
Application number	US-201815889990-A
Country	US
Kind code	B2
Filing date	Feb 6, 2018
Priority date	Aug 6, 2015
Publication date	Apr 5, 2022
Grant date	Apr 5, 2022

Abstract

The present invention provides a means to modulate gene expression in vivo in a manner that avoids problems associated with CRISPR endogenous protein knock-out or knock-in strategies and strategies that provide for correction, or alteration, of single nucleotides. The invention includes inserting into the genome a nucleotide encoding a heterobifunctional compound targeting protein (dTAG) in-frame with the nucleotide sequence of a gene encoding an endogenously expressed protein of interest which, upon expression, produces an endogenous protein-dTAG hybrid protein. This allows for targeted protein degradation of the dTAG and the fused endogenous protein using a heterobifunctional compound.

First claim

We claim:

1. A composition comprising an isolated cell and a heterobifunctional compound,
wherein the isolated cell comprises:
a nucleic acid sequence encoding a heterobifunctional compound targeting protein (dTAG) that has the amino acid sequence of SEQ ID NO: 1, wherein said dTAG is capable of being bound by a heterobifunctional compound selected from dFKBP-1-dFKBP-5, and a nucleic acid sequence encoding a CRISPR RNA-guided endonuclease;
wherein the CRISPR RNA-guided endonuclease, upon being expressed, acts to genomically integrate the nucleic acid encoding the heterobifunctional compound targeting protein;
wherein the nucleic acid sequence encoding the dTAG is integrated genomically in-frame in a 5′ or 3′ orientation with a nucleic acid sequence of a gene encoding an endogenous protein;
wherein expression of the gene encoding the endogenous protein produces an endogenous protein-dTAG hybrid protein;
wherein the heterobifunctional compound binds to a) the endogenous protein-dTAG hybrid protein through the dTAG and b) a ubiquitin ligase in a manner that brings the endogenous protein-dTAG hybrid protein into proximity of the ubiquitin ligase; and
wherein the endogenous protein-dTAG hybrid protein is ubiquitinated and then degraded by a proteasome: and
the heterobifunctional compound which is selected from dFKBP-1-dFKBP-5.

2. The composition of claim 1 , wherein the cell is a human cell.

3. The composition of claim 1 , wherein the nucleic acid sequence encoding the heterobifunctional compound targeting protein is inserted in frame with a gene encoding an endogenous protein associated with a disease that is a result of a gain of function mutation, amplification or increased expression, a monogenetic disease, a proteopathy, or a combination thereof.

4. The composition of claim 1 , wherein the CRISPR RNA-guided endonuclease is selected from Cas1, Cas IB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cpf1.

5. The composition of claim 4 , wherein the CRISPR RNA-guided endonuclease is a Cas9 endonuclease comprising an amino acid sequence of SEQ ID NO: 52.

6. The composition of claim 1 , wherein the heterobifunctional compound targeting protein does not interfere with the function of the endogenously expressed protein.

7. A method of reducing protein overexpression in a subject comprising:
transforming one or more cells of the subject with a nucleic acid sequence encoding a heterobifunctional compound targeting protein (dTAG), that has the amino acid sequence of SEQ ID NO: 1, wherein said dTAG is capable of being bound by a heterobifunctional compound selected from dFKBP-1-dFKBP-5;
wherein the nucleic acid sequence is integrated genomically in-frame in a 5′ or 3′ orientation with a nucleic acid sequence of an endogenous protein associated with a disease due to overexpression of the endogenous protein;
wherein insertion of the nucleic acid encoding the dTAG into the genomic sequence results in an endogenous protein-dTAG hybrid protein upon expression; and
administering to the subject a heterobifunctional compound;
wherein the heterobifunctional compound binds to a) the endogenous protein-dTAG hybrid protein through the dTAG and b) a ubiquitin ligase in a manner that brings the endogenous protein-dTAG hybrid into proximity of the ubiquitin ligase, wherein the endogenous protein-dTAG hybrid protein is ubiquitinated and then degraded by a proteasome; and

wherein the heterobifunctional compound is selected from dFKBP-1-dFKBP-5.

8. The method of claim 7 , wherein the subject is a human.

9. The method of claim 7 , wherein the nucleic acid sequence encoding the heterobifunctional compound targeting protein is inserted in frame with a gene encoding an endogenous protein associated with a disease that is a result of a gain of function mutation, amplification or increased expression, a monogenetic disease, a proteopathy, or a combination thereof.

10. The method of claim 7 , further comprising transforming one or more cells of the subject with a nucleic acid sequence encoding a CRISPR RNA-guided endonuclease, wherein upon expression of the nucleic acid sequence, the CRISPR RNA guided nuclease acts to genomically integrate the nucleic acid sequence encoding the heterobifunctional compound targeting protein.

11. The method of claim 10 , wherein the CRISPR RNA-guided endonuclease is selected from Cas1, Cas IB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cpf1.

12. The method of claim 7 , wherein the heterobifunctional compound targeting protein does not interfere with the function of the endogenously expressed protein.

13. An isolated cell, comprising:
a nucleic acid sequence encoding a heterobifunctional compound targeting protein (dTAG) that has the amino acid sequence of SEQ ID NO: 1, wherein said dTAG is capable of being bound by a heterobifunctional compound selected from dFKBP-1-dFKBP-5, and a nucleic acid sequence encoding a CRISPR RNA-guided endonuclease;
wherein the CRISPR RNA-guided endonuclease, upon being expressed, acts to genomically integrate the nucleic acid encoding the heterobifunctional compound targeting protein;
wherein the nucleic acid sequence encoding the dTAG is integrated genomically in-frame in a 5′ or 3′ orientation with a nucleic acid sequence of a gene encoding an endogenous protein;
wherein expression of the gene encoding the endogenous protein produces an endogenous protein-dTAG hybrid protein;
wherein the heterobifunctional compound binds to a) the endogenous protein-dTAG hybrid protein through the dTAG and b) a ubiquitin ligase in a manner that brings the endogenous protein-dTAG hybrid protein into proximity of the ubiquitin ligase; and
wherein the endogenous protein-dTAG hybrid protein is ubiquitinated and then degraded by a proteasome,
wherein the isolated cell further comprises the heterobifunctional compound; and
wherein the heterobifunctional compound is selected from dFKBP-1-dFKBP-5.

Assignees

• Dana Farber Cancer Inst Inc

Inventors

• Buckley Dennis
• Winter Georg
• Phillips Andrew J
• Heffernan Timothy
• Bradner James
• Roberts Justin
• Nabet Behnam

Classifications

• A61K40/4211

  CD19 or B4 · CPC title

• A61K40/4205

  Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4 · 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

• C07K2319/03

  containing a transmembrane segment · CPC title