Fusion protein for crispr/cas system and complex comprising the same and uses thereof

Patent metadata

Field	Value
Publication number	US-2019106688-A1
Application number	US-201816156864-A
Country	US
Kind code	A1
Filing date	Oct 10, 2018
Priority date	Oct 11, 2017
Publication date	Apr 11, 2019
Grant date	—

Abstract

Provided are a fusion protein used in a CRISPR/Cas system, a complex including the same, and uses thereof. The fusion protein may efficiently be used as an anticancer agent due to complexation with a guide RNA, remarkable intracellular delivery activity of the guide RNA in vivo or in vitro without any other cationic polymers or lipid carriers, and synergistic effects by co-administration with any other anticancer agent as well as anticancer activity by single treatment.

First claim

What is claimed is:

1 . A fusion protein comprising:
a CRISPR-associated protein (Cas protein); a nuclear localization sequence (NLS); and a cationic cell penetrating peptide.

2 . The fusion protein of claim 1 , wherein the Cas protein is a Cas9 protein.

3 . The fusion protein of claim 1 , wherein, the cationic cell penetrating peptide is a low molecular weight protamine (LMWP).

4 . The fusion protein of claim 1 , wherein the NLS binds to a C-terminal of the Cas protein, and the cationic cell penetrating peptide binds to a C-terminal of the NLS.

5 . A host cell transformed by an expression vector including a nucleic acid molecule encoding the fusion protein according to claim 1 .

6 . The host cell of claim 5 , wherein the cell is selected from group consisting of a cell of yeast, fungus, protozoa, plant, insect, amphibian and mammalian.

7 . A complex comprising:
a fusion protein comprising a CRISPR-associated protein (Cas protein) and a cationic cell penetrating peptide; and a guide RNA.

8 . The complex of claim 7 , wherein the guide RNA is a dual RNA comprising a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA), or a single-stranded guide RNA (sgRNA) comprising portions of the crRNA and the tracrRNA and hybridizing with a target DNA.

9 . The complex of claim 8 , wherein the crRNA binds to the tracrRNA.

10 . The complex of claim 7 , wherein the complex targets a sequence of KRAS represented by SEQ ID NO: 10.

11 . The complex of claim 7 , wherein the fusion protein further comprises a nuclear localization sequence (NLS).

12 . The complex of claim 7 , wherein the complex is self-assembled by the fusion protein to form a complex with a guide RNA.

13 . A method of preparing a complex to deliver a guide RNA into a cell including brining the fusion protein according to claim 1 into contact with the guide RNA.

14 . A method of treating cancer, the method including administering a pharmaceutical composition comprising the complex according to claim 8 into an individual in need thereof.

15 . The method of claim 14 , the pharmaceutical composition further comprising a targeted anticancer agent.

16 . The method of claim 14 , wherein the cancer is selected from lung cancer, non-small-cell lung cancer, pancreatic cancer, gastric cancer, liver cancer, colon cancer, brain cancer, breast cancer, thyroid cancer, bladder cancer, esophageal cancer, or uterine cancer.

17 . The method of claim 14 , wherein the complex targets a sequence of KRAS represented by SEQ ID NO: 9.

Assignees

• Korea Inst Sci & Tech

Inventors

• Oh Seung Ja
• Jang Mi Hue
• Kim Seung Min

Classifications

• A61K45/06

  Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca · CPC title

• C12N2800/80

  Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites · CPC title

• A61K38/465

  acting on ester bonds (3.1), e.g. lipases, ribonucleases · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title