# US 10,308,960 B2 · Methods for treating cells containing fusion genes

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- Number: US 10,308,960 B2
- Document ID: US10308960B2
- Status: Granted
- Kind: B2
- Application number: 15/406,472
- Filed: 2017-01-13
- Published: 2017-08-24
- Granted: 2019-06-04
- Earliest claimed: 2014-07-17
- Assignees: University of Pittsburgh—Of the Commonwealth System of Higher Education
- Applicants: University of Pittsburgh—of the Commonwealth System of Higher Education
- Inventors: Jianhua Luo; Yanping Yu; Zhanghui Chen; George Konstantine Michalopoulos; Joel Nelson
- Web page: https://patentgrep.com/patent/US10308960B2

## CPC

- C12N 15/907: {in mammalian cells}
- A61K 31/522: having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
- A61K 38/1761: {Apoptosis related proteins, e.g. Apoptotic protease-activating factor-1 (APAF-1), Bax, Bax-inhibitory protein(s)(BI; bax-I), Myeloid cell leukemia associated protein (MCL-1), Inhibitor of apoptosis [IAP] or Bcl-2}
- A61K 48/0058: {Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct}
- A61P 35/00: Antineoplastic agents
- C12N 9/1211: {Thymidine kinase (2.7.1.21)}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575)
- C12Y 207/01021: Thymidine kinase (2.7.1.21)
- C12N 2310/10: Type of nucleic acid
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12Q 2600/156: Polymorphic or mutational markers
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Y: ENZYMES

## Abstract

The present invention relates to methods for treating prostate cancer patients. It is based, at least in part, on the discovery that approximately 90% of men carrying at least one of the following fusion genes: TRMT11-GRIK2, SLC45A2-AMACR, MTOR-TP53BP1, LRRC59-FLJ60017, TMEM135-CCDC67 and CCNH-05orf30 experienced prostate cancer recurrence, metastases and/or prostate cancer-specific death after radical prostatectomy (each examples of “progressive prostate cancer”), while these outcomes occurred in only 36% of men not carrying any of these fusion genes. It is also based, at least in part, on the discovery that a genome editing technique that specifically targets a fusion gene can induce cell death in a cancer cell that carries the fusion gene.

## Claims

### Claim 1

A method of treating a subject having prostate cancer, comprising determining whether a sample of the subject contains one or more fusion genes selected from the group consisting of TRMT11-GRIK2, SLC45A2-AMACR, MTOR-TP53BP1, LRRC59-FLJ60017, TMEM135-CCDC67, KDM4B-AC011523.2, MAN2A1-FER, PTEN-NOLC1, CCNH-C5orf30, ZMPSTE24-ZMYM4, CLTC-ETV1, ACPP-SEC13, DOCK7-OLR1, PCMTD1-SNTG1 and a combination thereof; and where if one or more fusion genes are detected in the sample then performing a CRISPR/Cas genome editing technique targeting the one or more fusion genes present within one or more prostate cancer cells of the subject,
wherein the genome editing technique comprises introducing into the one or more prostate cancer cells: (i) a vector comprising a nucleic acid encoding a Cas protein or a mutant thereof and two guide RNAs (gRNA), wherein one gRNA is complementary to a region within one gene of the fusion gene and a second gRNA is complementary to a region within a second gene of the fusion gene, and (ii) a vector comprising a donor nucleic acid and one or more targeting sequences,
wherein the two gRNAs direct binding of the Cas 9 protein or mutant thereof to a genomic sequence of the fusion gene to promote cleavage of the fusion gene and insertion of the donor nucleic acid into the fusion gene, and wherein expression of the donor nucleic acid induces cell death of the one or more prostate cancer cells.

### Claim 2 (depends on 1)

The method of claim 1, wherein the fusion gene is TMEM135-CCDC67 or CCNH-C5orf30.

### Claim 3 (depends on 1)

The method of claim 1, wherein the fusion gene is detected by FISH analysis.

### Claim 4 (depends on 1)

The method of claim 1, wherein the fusion gene is detected by reverse transcription polymerase chain reaction.

### Claim 5 (depends on 1)

The method of claim 1, wherein the donor nucleic acid encodes HSV-1 thymidine kinase.

### Claim 6 (depends on 1)

The method of claim 1 further comprising administering to the subject a therapeutically effective amount of ganciclovir or valganciclovir.

### Claim 7 (depends on 1)

The method of claim 1, wherein the one or more targeting sequences are complementary to one or more sequences of the fusion gene to promote homologous recombination of and the insertion of the donor nucleic acid into the fusion gene.

### Claim 8 (depends on 1)

The method of claim 1, wherein the Cas 9 protein is Cas 9D10A.