# US 11,078,487 B2 · Cancer-specific trans-splicing ribozymes and use thereof

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- Number: US 11,078,487 B2
- Document ID: US11078487B2
- Status: Granted
- Kind: B2
- Application number: 16/797,663
- Filed: 2020-02-21
- Published: 2020-08-27
- Granted: 2021-08-03
- Earliest claimed: 2019-02-22
- Assignees: Rznomics Inc.; Dong-A University Research Foundation for Industry-Academy Cooperation; Industry-Academic Cooperation Foundation, Dankook University
- Applicants: Rznomics Inc.; Dong-A University Research Foundation for Industry Academy Cooperation; Industry-Academic Cooperation Foundation, Dankook University
- Inventors: Seong-Wook Lee; Chang Ho Lee; Seung Ryul I Han; Ji Hyun Kim; Eun Yi Cho; Jin Sook Jeong; Mi Ha Ju
- Web page: https://patentgrep.com/patent/US11078487B2

## CPC

- C12N 15/1135: {against oncogenes or tumor suppressor genes}
- C12N 15/1137: {against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138)}
- 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 15/86: Viral vectors
- A01K 2207/12: Animals modified by administration of exogenous cells
- A01K 2227/105: Murine
- A01K 2267/0331: Animal model for proliferative diseases
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- C12N 2310/124: based on group I or II introns
- C12N 2310/141: MicroRNAs, miRNAs
- C12N 2320/32: Special delivery means, e.g. tissue-specific
- C12N 2710/10343: 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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)

## Abstract

A cancer-specific trans-splicing ribozyme and a use thereof are disclosed. The trans-splicing ribozyme does not act on normal tissue, but is specifically expressed in cancer tissue. Therefore, it is very safe and has excellent expression efficiency at the post-transcription level, and thus can be effectively used in treatment of cancer.

## Claims

### Claim 1

A nucleic acid construct, comprising, in the order from 5′ end to 3′ end:
(i) a cytomegalovirus (CMV) promoter;
(ii) a splicing donor/splicing acceptor sequence (SD/SA sequence);
(iii) a ribozyme-desired gene expression cassette, which is operably linked to the CMV promoter (i), includes (a) a sequence encoding a trans-splicing ribozyme targeting a cancer-specific gene and (b) a target gene connected to a 3′ exon of the ribozyme-encoding sequence;
(iv) a Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE); and
(v) a nucleic acid sequence complementary to microRNA-122 (miR-122), said nucleic acid sequence (v) being connected to the 3′ end of the WPRE and comprising one or more copies of the nucleic acid sequence of SEQ ID NO: 5,
wherein the SD/SA sequence is connected to the 5′ end of the ribozyme-desired gene expression cassette, and the WPRE is connected to the 3′ end of the ribozyme-desired gene expression cassette, and
wherein the cancer-specific gene is a telomerase reverse transcriptase (TERT) mRNA sequence.

### Claim 2 (depends on 1)

The nucleic acid construct of claim 1, wherein the TERT mRNA sequence comprises the nucleic acid sequence of SEQ ID NO: 2.

### Claim 3 (depends on 1)

The nucleic acid construct of claim 1, wherein the trans-splicing ribozyme comprises the nucleic acid sequence of SEQ ID NO: 3.

### Claim 4 (depends on 1)

The nucleic acid construct of claim 1, wherein the desired gene is an anti-cancer therapeutic gene or a reporter gene.

### Claim 5 (depends on 4)

The nucleic acid construct of claim 4, wherein the anti-cancer therapeutic gene is selected from the group consisting of a drug-sensitizing gene, a proapoptotic gene, a cytostatic gene, a cytotoxic gene, a tumor suppressor gene, an antigenic gene, a cytokine gene and an anti-angiogenic gene.

### Claim 6 (depends on 5)

The nucleic acid construct of claim 5, wherein the drug-sensitizing gene is a herpes simplex virus thymidine kinase (HSVtk) gene.

### Claim 7 (depends on 6)

The nucleic acid construct of claim 6, wherein the HSVtk gene comprises the nucleic acid sequence of SEQ ID NO: 4.

### Claim 8 (depends on 4)

The nucleic acid construct of claim 4, wherein the reporter gene is luciferase, a green fluorescent protein (GFP), a modified green fluorescent protein (mGFP), an enhanced green fluorescent protein (EGFP), a red fluorescent protein (RFP), a modified red fluorescent protein (mRFP), an enhanced red fluorescent protein (ERFP), a blue fluorescent protein (BFP), an enhanced blue fluorescent protein (EBFP), a yellow fluorescent protein (YFP), an enhanced yellow fluorescent protein (EYFP), a cyan fluorescent protein (CFP) or an enhanced cyan fluorescent protein (ECFP).

### Claim 9 (depends on 1)

A gene delivery system comprising the nucleic acid construct of claim 1.

### Claim 10 (depends on 1)

A ribozyme expressed from the nucleic acid construct of claim 1.

### Claim 11 (depends on 1)

A pharmaceutical composition, comprising the nucleic acid construct of claim 1, a gene delivery system comprising the nucleic acid construct, or a ribozyme expressed from the nucleic acid construct as an active ingredient.

### Claim 12 (depends on 11)

The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is in a formulation suitable for administration by intravenous, intraartery, intratumor, or subcutaneous route.

### Claim 13 (depends on 11)

The pharmaceutical composition of claim 11, wherein the pharmaceutical composition is in a formulation suitable for injection or infusion.

### Claim 14 (depends on 1)

A method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition comprising the nucleic acid construct claim 1, a gene delivery system comprising the nucleic acid construct, or a ribozyme expressed from the nucleic acid construct as an active ingredient,
wherein the cancer is liver cancer or glioblastoma.

### Claim 15 (depends on 14)

The method of claim 14, wherein, in cancer tissue of the subject, a copy number of miR-122 expressed in a cancer tissue is less than 100 times a copy number of ribozymes expressed from the nucleic acid construct in the cancer tissue.

### Claim 16 (depends on 14)

The method of claim 14, wherein, in cancer tissue of the subject, miR-122 is substantially not expressed in cancer tissue.

### Claim 17 (depends on 14)

The method of claim 14, wherein the liver cancer is caused by any one or more selected from the group consisting of hepatitis B virus, hepatitis C virus decreasing miR-122 expression in liver cancer tissue, alcohol, chronic hepatitis, liver cirrhosis, non-alcoholic fatty acid disease, aflatoxin, and family history.