Cancer specific-splicing ribozyme and use thereof
Inventors
Lee, Seong Wook • Kim, Ju Hyun • Jeong, Jin Sook • HAN, Sang Young
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-10280420-B2
Publication Date
2019-05-07
Expiration Date
Abstract
The present invention relates to a recombination vector, a transformation cell into which the recombinant vector is introduced, a ribozyme expressed from the recombination vector, a prophylactic or therapeutic composition for liver cancer comprising the recombination vector and the ribozyme, and a therapeutic method for liver cancer using the composition, said recombination vector comprising: a tissue-specific promoter; and a ribozyme-target gene expression cassette comprising a trans-splicing ribozyme targeting a cancer-specific gene and a target gene connected to the 3′ exon of the ribozyme, wherein a splicing donor/splicing acceptor sequence (SD/SA sequence) is connected to the 5′ end of the ribozyme-target gene expression cassette, woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is connected to the 3′ end of the ribozyme-target gene expression cassette, and a nucleic acid sequence recognizing a micro RNA-122a (microRNA-122a, miR-122a) is further connected to the 3′ end of the WPRE.
Core Innovation
The invention describes a recombinant vector comprising a phosphoenolpyruvate carboxykinase (PEPCK) promoter as a liver cell-specific promoter comprising a nucleic acid sequence of SEQ ID NO: 2. The vector further comprises a ribozyme-target gene expression cassette that uses a telomerase reverse transcriptase (TERT) mRNA as a trans-splicing ribozyme targeting a cancer-specific gene. A herpes simplex virus-thymidine kinase (HSVtk) gene is used as a target gene linked to the 3′ exon of the ribozyme.
A splicing donor/splicing acceptor sequence (SD/SA sequence) is linked to the 5′ end of the ribozyme-target gene expression cassette, and a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is linked to the 3′ end of the ribozyme-target gene expression cassette. The vector additionally includes a nucleic acid sequence that binds complementarily to micro RNA-122a (microRNA-122a, miR-122a) further linked to the 3′ end of the WPRE.
The disclosed therapeutic embodiments include transformed cells having the recombinant vector, with ribozyme expression driven by the vector. The disclosure also provides a pharmaceutical composition comprising the recombinant vector and therapeutic methods involving administering a ribozyme to a subject in need for treating hepatocellular carcinoma.
Claims Coverage
The document includes one independent claim focused on a recombinant vector, with dependent claims that specify sequence identities and extend coverage to transformed cells, pharmaceutical compositions, and treatment methods. The main coverage rests on combining a liver cell-specific PEPCK promoter with a trans-splicing ribozyme cassette targeting cancer-specific gene expression, together with SD/SA and WPRE regulatory elements and an miR-122a complementary binding sequence at the 3′ end of WPRE.
Liver cell-specific PEPCK promoter driving a trans-splicing ribozyme targeting a cancer-specific gene via TERT mRNA
A recombinant vector comprising a phosphoenolpyruvate carboxykinase (PEPCK) promoter as a liver cell-specific promoter comprising a nucleic acid sequence of SEQ ID NO: 2, and a ribozyme-target gene expression cassette comprising a telomerase reverse transcriptase (TERT) mRNA as a trans-splicing ribozyme targeting a cancer-specific gene.
HSVtk target gene linked to the ribozyme 3′ exon
The ribozyme-target gene expression cassette includes a herpes simplex virus-thymidine kinase (HSVtk) gene as a target gene linked to the 3′ exon of the ribozyme.
SD/SA linked to the 5′ end and WPRE linked to the 3′ end of the ribozyme-target cassette
A splicing donor/splicing acceptor sequence (SD/SA sequence) is linked to the 5′ end of the ribozyme-target gene expression cassette, and a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is linked to the 3′ end of the ribozyme-target gene expression cassette.
miR-122a complementary binding sequence linked to the 3′ end of WPRE
A nucleic acid sequence that binds complementarily to micro RNA-122a (miR-122a) is further linked to the 3′ end of the WPRE.
Specific trans-splicing ribozyme nucleic acid sequence
The recombinant vector of claim 1 is such that the trans-splicing ribozyme contains the nucleic acid sequence of SEQ ID NO: 5.
Specific HSVtk gene nucleic acid sequence
The recombinant vector of claim 1 is such that the HSVtk gene includes the nucleic acid sequence identified as SEQ ID NO: 6.
Transformed cell comprising the recombinant vector
A transformed cell is provided in which the recombinant vector of claim 1 has been introduced.
Pharmaceutical composition including the recombinant vector as active ingredient for preventing or treating hepatocellular carcinoma
A pharmaceutical composition includes the recombinant vector of claim 1 as an active ingredient for preventing or treating hepatocellular carcinoma.
Method of treating hepatocellular carcinoma by administering a pharmaceutically effective amount of a ribozyme
A method to treat hepatocellular carcinoma by administering a pharmaceutically effective amount of the ribozyme of claim 5 to a subject in need.
Overall, the independent claim centers on a recombinant vector that combines a PEPCK liver cell-specific promoter with a TERT mRNA-based trans-splicing ribozyme targeting a cancer-specific gene, includes HSVtk linked to the 3′ exon of the ribozyme, and incorporates SD/SA and WPRE elements with an miR-122a complementary sequence linked at the 3′ end of WPRE. Dependent claims specify SEQ ID NO: 5 for the trans-splicing ribozyme, SEQ ID NO: 6 for HSVtk, and extend the scope to transformed cells, pharmaceutical compositions, and treatment by administering a ribozyme.
Stated Advantages
Increases ribozyme expression efficiency.
Improves cancer-cell selectivity.
Reduces toxicity to normal liver.
Reduces tumor burden with minimal hepatotoxicity.
Enables dose reduction compared with a control vector.
Documented Applications
Preventing or treating hepatocellular carcinoma using a pharmaceutical composition comprising the recombinant vector.
Treating hepatocellular carcinoma by administering a pharmaceutically effective amount of the ribozyme to a subject in need.
Interested in licensing this patent?