IMiD screening methods IMiD-sensitive cells with mutant CRBN

Patent metadata

Field	Value
Publication number	US-10334829-B2
Application number	US-201815955073-A
Country	US
Kind code	B2
Filing date	Apr 17, 2018
Priority date	Nov 8, 2013
Publication date	Jul 2, 2019
Grant date	Jul 2, 2019

Abstract

The present invention features a knock-in mouse comprising a mutation in an endogenous CRBN locus and methods of use thereof.

First claim

What is claimed is:

1. A method of screening for an immunomodulatory imide drug (IMiD) that activates ubiquitin ligase, the method comprising:
a) contacting an isolated mouse cell with an IMiD, wherein the genome of the cell comprises an exogenous polynucleotide encoding a mutant cereblon (CRBN) polypeptide at the endogenous CRBN locus, wherein said mutant CRBN comprises the point mutation I391V, and wherein the cell has IMiD sensitivity; and
b) detecting the amount of:
i) ubiquitination of IKAROS family zinc finger protein 1 (IKZF1) or IKAROS family zinc finger protein 3 (IKZF3);
ii) degradation of IKZF1 or IKZF3; or
iii) binding of CRBN to IKZF1 or IKZF3,

in the isolated cell obtained in step a);
wherein increased ubiquitination or degradation of IKZF1 or IKZF3 or increased binding of CRBN to IKZF1 or IKZF3 as compared to a wild-type isolated mouse cell indicates the IMiD activates ubiquitin ligase.

2. The method of claim 1 , further comprising detecting:
i) ubiquitination of the proteome; or
ii) total protein level in the proteome, in the isolated cell of step a).

3. The method of claim 1 , wherein the immunomodulatory imide drug (IMiD) or agent is lenalidomide or an analog thereof.

4. The method of claim 3 , wherein the analog of lenalidomide is thalidomide or pomalidomide.

5. A method of screening for an immunomodulatory imide drug (IMiD) that activates ubiquitin ligase, the method comprising:
a) contacting an isolated mouse cell with an IMiD, wherein the genome of the cell comprises an exogenous polynucleotide encoding a mutant cereblon (CRBN) polypeptide at the endogenous CRBN locus, wherein said mutant CRBN comprises a point mutation selected from the group consisting of S369C, V380E, and I391V, and wherein the cell has IMiD sensitivity; and
b) detecting the amount of:
i) ubiquitination of IKAROS family zinc finger protein 1 (IKZF1) or IKAROS family zinc finger protein 3 (IKZF3);
ii) degradation of IKZF1 or IKZF3; or
iii) binding of CRBN to IKZF1 or IKZF3,

in the isolated cell obtained in step a);
wherein increased ubiquitination or degradation of IKZF1 or IKZF3 or increased binding of CRBN to IKZF1 or IKZF3 as compared to a wild-type isolated mouse cell indicates the IMiD activates ubiquitin ligase.

6. The method of claim 5 , further comprising detecting:
i) ubiquitination of the proteome; or
ii) total protein level in the proteome, in the isolated cell of step a).

7. The method of claim 5 , wherein the immunomodulatory imide drug (IMiD) or agent is lenalidomide or an analog thereof.

8. The method of claim 7 , wherein the analog of lenalidomide is thalidomide or pomalidomide.

9. An isolated mouse cell whose genome comprises an exogenous polynucleotide encoding a mutant cereblon (CRBN) polypeptide at the endogenous CRBN locus, wherein said mutant CRBN comprises a point mutation selected from the group consisting of S369C, V380E, and I391V, and wherein the cell has immunomodulatory imide drug (IMiD) sensitivity.

Assignees

• Broad Inst Inc
• Brigham & Womens Hospital Inc

Inventors

• Ebert Benjamin Levine
• Krönke Jan
• Carr Steven A
• Udeshi Namrata D
• Fink Emma

Classifications

• G01N33/57595

  involving intracellular compounds · CPC title

• C12Q2600/158

  Expression markers · CPC title

• C12Q2600/106

  Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism · CPC title

• C12Q2600/156

  Polymorphic or mutational markers · CPC title

• A01K67/0278 Primary

  Knock-in vertebrates, e.g. humanised vertebrates · CPC title