# US 9,434,949 B2 · Uses of the human ZFX gene and drugs associated with same

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 9,434,949 B2
- Document ID: US9434949B2
- Status: Granted
- Kind: B2
- Application number: 14/239,120
- Filed: 2011-08-16
- Published: 2014-09-18
- Granted: 2016-09-06
- Earliest claimed: 2011-08-16
- Assignees: Shanghai Genechem Co., Ltd.
- Applicants: Yueqiong Cao; Dalong Lv; Xiangying Zhu; Haixiong Han; Honghua Qu; Yangsheng Jin
- Inventors: Yueqiong Cao; Dalong Lv; Xiangying Zhu; Haixiong Han; Honghua Qu; Yangsheng Jin
- Web page: https://patentgrep.com/patent/US9434949B2

## CPC

- C12N 15/1135: {against oncogenes or tumor suppressor genes}
- A61K 31/7105: Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
- A61K 31/713: Double-stranded nucleic acids or oligonucleotides
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 35/00: Antineoplastic agents
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- 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

## Abstract

The present invention discloses uses of the human ZFX gene and drugs associated therewith. Also disclosed are uses of the human ZFX gene in tumor treatment, tumor diagnosis, and drug preparation. Further disclosed are small interfering RNA (siRNA), and nucleic acid and lentivirus encoding the siRNA to the human ZFX gene and uses thereof. The siRNA and nucleic acid and lentivirus encoding the siRNA provided by the present invention can specifically inhibit the expression of human ZFX gene. Lentiviruses in particular can efficiently infect target cells, inhibit ZFX expression in target cells, and inhibit the growth of tumor cells, thus promote tumor apoptosis and have great significance in tumor treatment.

## Claims

### Claim 1

A method for inhibiting or attenuating the growth, proliferation, and/or survival of a tumor cell, which method comprises administering to the tumor cell a nucleic acid that specifically inhibits transcription or translation of a zinc finger protein X-linked (ZFX) gene, or expression of the ZFX protein, thereby inhibiting the growth, proliferation, and/or survival of the tumor cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the nucleic acid comprises an antisense oligonucleotide, a double stranded RNA (dsRNA), a ribozyme, an RNase III-prepared short interfering RNA (esiRNA) or a vector-expressed short hairpin RNAs (shRNA).

### Claim 3 (depends on 2)

The method of claim 2, wherein the dsRNA, ribozyme, esiRNA or shRNA comprises a subsequence of a promoter of the ZFX gene or a subsequence of the ZFX gene.

### Claim 4 (depends on 3)

The method of claim 3, wherein the dsRNA is an siRNA.

### Claim 5 (depends on 4)

The method of claim 4, wherein the siRNA comprises a sense strand, which comprises a nucleotide sequence substantially identical to a target sequence in the ZFX gene, and an antisense strand, wherein the sense and antisense strands form an RNA duplex.

### Claim 6 (depends on 1)

The method of claim 1, wherein the ZFX gene is from human.

### Claim 7 (depends on 5)

The method of claim 5, wherein the target sequence in the ZFX gene comprises a sequence set forth in SEQ ID NOs: 1-42.

### Claim 8 (depends on 1)

The method of claim 1, wherein the tumor is selected from the group consisting of laryngeal cancer, lung cancer, stomach cancer, liver cancer, mammary gland cancer and glioma.

### Claim 9 (depends on 1)

The method of claim 1, wherein the growth of the tumor cell is associated with the expression of the ZFX protein.

### Claim 10 (depends on 1)

The method of claim 1, wherein the nucleic acid is administered at a level sufficient to lower the transcription or translation of the ZFX gene, or the expression of the ZFX protein.

### Claim 11 (depends on 10)

The method of claim 10, wherein the expression of the ZFX gene is attenuated by at least about 50%, 80%, 90%, 95% or 99%.

### Claim 12 (depends on 1)

The method of claim 1, wherein the nucleic acid comprises an oligonucleotide that attenuates the expression of the ZFX gene in the tumor cell, which oligonucleotide comprises:
a) a dsRNA sequence comprising a nucleotide sequence that hybridizes under stringent conditions to the ZFX gene; or
b) a DNA sequence comprising a nucleotide sequence that is transcribed in the tumor cell to an shRNA comprising a nucleotide sequence that hybridizes under stringent conditions to the ZFX gene.

### Claim 13 (depends on 12)

The method of claim 12, wherein a first strand of the dsRNA is substantially identical to about 15-27 consecutive nucleotides of the ZFX gene, and a second strand of the dsRNA is substantially complementary to the first strand.

### Claim 14 (depends on 12)

The method of claim 12, wherein a first strand of the dsRNA is substantially identical to a target sequence in the ZFX gene.

### Claim 15 (depends on 12)

The method of claim 12, wherein the ZFX gene is from human.

### Claim 16 (depends on 12)

The method of claim 12, wherein the ZFX gene comprises a sequence set forth in SEQ ID NO: 1.

### Claim 17 (depends on 14)

The method of claim 14, wherein the target sequence in the ZFX gene comprises a sequence set forth in SEQ ID NOs: 1-42.

### Claim 18 (depends on 12)

The method of claim 12, wherein the shRNA comprises a sense fragment, which comprises a nucleotide sequence substantially identical to a target sequence in the ZFX gene, and an antisense fragment, wherein the sense and antisense fragments are separated by a loop fragment.

### Claim 19 (depends on 18)

The method of claim 18, wherein the loop fragments comprises a sequence selected from the group consisting of UUCAAGAGA, AUG, CCC, UUCG, CCACC, CTCGAG, AAGCUU and CCACACC.

### Claim 20 (depends on 19)

The method of claim 19, wherein the shRNA comprises the sequence set forth in SEQ ID NO: 43.

### Claim 21 (depends on 1)

The method of claim 1, wherein the nucleic acid comprises a lentivirus vector comprising a DNA sequence comprising a nucleotide sequence that is transcribed in the tumor cell to an shRNA comprising a nucleotide sequence that hybridizes under stringent conditions to the ZFX gene,
wherein the shRNA comprises a sense fragment, which comprises a nucleotide sequence substantially identical to a target sequence in the ZFX gene, and an antisense fragment, wherein the sense and antisense fragments are separated by a loop fragment.

### Claim 22 (depends on 21)

The method of claim 21, wherein the lentivirus vector further comprises a promoter sequence.

### Claim 23 (depends on 21)

The method of claim 21, wherein the lentivirus vector further comprises a nucleotide sequence encoding a detectable marker in the tumor cell.

### Claim 24 (depends on 23)

The method of claim 23, wherein the detectable marker is a green fluorescent protein (GFP).

### Claim 25 (depends on 21)

The method of claim 21, wherein the lentivirus vector is selected from the group consisting of pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP, pLKO.1-CMV-Neo, pLKO.1-Neo, pLKO.1-Neo-CMV-tGFP, pLKO.1-puro-CMV-TagCFP, pLKO.1-puro-CMV-TagYFP, pLKO.1-puro-CMV-TagRFP, pLKO.1-puro-CMV-TagFP635, pLKO.1-puro-UbC-TurboGFP, pLKO.1-puro-UbC-TagFP635, pLKO-puro-IPTG-1xLacO, pLKO-puro-IPTG-3xLacO, pLP1, pLP2, pLP/VSV-G, pENTR/U6, pLenti6/BLOCK-iT-DEST, pLenti6-GW/U6-laminshrna, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP and pLenti6.2/N-Lumio/V5-GW/lacZ.

### Claim 26

A method for treating tumor in a subject, which method comprises administering an effective amount of a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises an oligonucleotide that attenuates the expression of a zinc finger protein X-linked (ZFX) gene in a tumor cell, which oligonucleotide comprises:
a) a dsRNA sequence comprising a nucleotide sequence that hybridizes under stringent conditions to the ZFX gene; or
b) a DNA sequence comprising a nucleotide sequence that is transcribed in the tumor cell to an shRNA comprising a nucleotide sequence that hybridizes under stringent conditions to the ZFX gene.

### Claim 27 (depends on 1)

The method of claim 1, wherein the nucleic acid comprises a fragment of the ZFX gene, wherein the nucleic acid is not the full-length nucleotide sequence of the ZFX gene.