Cancer-specific trans-splicing ribozymes and use thereof
Inventors
LEE, SEONG-WOOK • Lee, Chang Ho • Han, Seung Ryul I • Kim, Ji Hyun • Cho, Eun Yi • Jeong, Jin Sook • Ju, Mi Ha
Assignees
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Publication Number
US-11078487-B2
Publication Date
2021-08-03
Expiration Date
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.
Core Innovation
The invention provides a nucleic acid construct comprising a cytomegalovirus (CMV) promoter, a splicing donor/splicing acceptor sequence (SD/SA sequence), a ribozyme-desired gene expression cassette, and a Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). 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.
The ribozyme-desired gene expression cassette includes a sequence encoding a trans-splicing ribozyme targeting a cancer-specific gene and a target gene connected to a 3′ exon of the ribozyme-encoding sequence. The cancer-specific gene is a telomerase reverse transcriptase (TERT) mRNA sequence. The construct further includes a nucleic acid sequence complementary to microRNA-122 (miR-122), which is connected to the 3′ end of the WPRE and comprises one or more copies of the nucleic acid sequence of SEQ ID NO: 5.
The background problem addressed is the need for cancer-specific gene expression and activity of a trans-splicing ribozyme system while avoiding undesired activity in non-target tissues. The provided system uses miR-122 complementary sequences positioned with the WPRE to enable ribozyme regulation based on miR-122 levels, supporting anti-cancer expression where miR-122 is low and suppression where miR-122 is high.
The disclosed construct is supported by gene-delivery and pharmaceutical-composition concepts and described experiments indicating enhanced expression efficiency, selective killing across multiple cancers, and potential efficacy in sorafenib-resistant liver cancer. The document also associates miR-122-mediated regulation with reduced hepatotoxicity and includes in vivo distribution and toxicity readouts.
Claims Coverage
The partial content identifies one independent claim directed to the nucleic acid construct, with dependent claims refining sequence identities, miR-122/ribozyme relationships in cancer tissue, therapeutic use, administration routes, and gene delivery/pharmaceutical composition aspects.
CMV promoter-linked SD/SA trans-splicing ribozyme cassette
A nucleic acid construct with a cytomegalovirus (CMV) promoter and a splicing donor/splicing acceptor sequence (SD/SA sequence) connected to a ribozyme-desired gene expression cassette, where the cassette includes a sequence encoding a trans-splicing ribozyme.
TERT mRNA-targeting cancer-specific trans-splicing ribozyme
The trans-splicing ribozyme targets a cancer-specific gene, wherein the cancer-specific gene is a telomerase reverse transcriptase (TERT) mRNA sequence.
WPRE posttranscriptional regulatory element at cassette 3′ end
A Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) connected to the 3′ end of the ribozyme-desired gene expression cassette.
miR-122 complementary sequence with SEQ ID NO: 5 copies at WPRE 3′ end
A nucleic acid sequence complementary to microRNA-122 (miR-122), connected to the 3′ end of the WPRE and comprising one or more copies of the nucleic acid sequence of SEQ ID NO: 5.
Across the independent claim and its refinements shown in the partial content, coverage centers on a nucleic acid construct combining CMV promoter and SD/SA-dependent trans-splicing ribozyme expression targeting TERT mRNA, with WPRE positioning and miR-122-complementary regulation using SEQ ID NO: 5.
Stated Advantages
Enhanced expression efficiency.
Selective killing across multiple cancers.
Potential efficacy in sorafenib-resistant liver cancer.
Reduced hepatotoxicity associated with miR-122-mediated regulation.
Documented Applications
Treatment of liver cancer and glioblastoma in a subject by administering a pharmaceutical composition containing the nucleic acid construct.
Use in a gene delivery system that includes the nucleic acid construct.
Pharmaceutical compositions formulated for intravenous, intraartery, intratumor, or subcutaneous administration.
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