Recombinant oncolytic virus, preparation method therefor, use thereof and medicine thereof
View Patent ↗Provided are an oncolytic virus, a preparation method therefor, the use thereof and a medicine thereof, wherein the genome of the oncolytic virus includes the following exogenous elements: (1) a first expression cassette containing a first promoter and a first interfering RNA expression sequence; (2) a target sequence; and (3) a second expression cassette. The replication of the oncolytic virus is regulated and controlled by exogenous elements inserted into the genome sequence thereof; by means of the regulation and control by the exogenous elements, the oncolytic virus can be selectively replicated in different types of cells, and thus, second cells, that is, target cells (such as tumor cells), can be selectively killed, and first cells, that is, non-target cells (such as normal cells), are not damaged.
1 . An oncolytic virus, wherein the genome of the oncolytic virus contains the following exogenous elements:
(1) a first expression cassette comprising a first promoter, a first interfering RNA expression sequence for expressing a first interfering RNA, and a second interfering RNA expression sequence for expressing a second interfering RNA;
(2) a target sequence; and
(3) a second expression cassette,
wherein:
the nucleotide sequence of the target sequence is the sequence set forth in SEQ ID NO: 1, the nucleotide sequence of the first interfering RNA is the sequence set forth in SEQ ID NO: 2, and the nucleotide sequence of the second interfering RNA is the sequence set forth in SEQ ID NO: 3;
the first interfering RNA expression sequence is used to express the first interfering RNA which binds to the target sequence in a first cell, the first interfering RNA expression sequence being driven by the first promoter so as to express the first interfering RNA in the first cell;
the target sequence is located at the 5′ or 3′ untranslated region (UTR) of an essential gene required for replication of the oncolytic virus;
the second expression cassette comprises a second promoter and an inhibitory component expression sequence, wherein the inhibitory component expression sequence is used to express an inhibitory component which inhibits the biosynthesis and/or bioactivity of a first enzyme involved in the biosynthesis of interfering RNA, in a second cell, the inhibitory component expression sequence being driven by the second promoter so as to express the inhibitory component in the second cell, wherein the inhibitory component comprises a third interfering RNA which inhibits expression of a second enzyme involved in the biosynthesis of the first enzyme, wherein the nucleotide sequence of the third interfering RNA is the sequence set forth in SEQ ID NO:4; and
the first and the second cells are different cell types.
2 . The oncolytic virus of claim 1 , wherein the second interfering RNA acts on the open reading frame (ORF) of a nonessential gene, which is not needed for the virus to replicate in vitro, so as to interfere with expression of the nonessential gene, and the second interfering RNA expression sequence is expressed under the control of the first promoter.
3 . The oncolytic virus of claim 2 , wherein the second cell is a tumor cell of a mammal, and the first cell is a non-tumor cells of the mammal.
4 . The oncolytic virus of claim 1 , wherein the target sequence is inserted into the 5′ or 3′ UTR of one or more essential genes of the oncolytic virus.
5 . The oncolytic virus of claim 4 , wherein the oncolytic virus is selected from the group consisting of herpes simplex virus, adenovirus, vaccinia virus, Newcastle disease virus, poliovirus, coxsackie virus, measles virus, mumps virus, vaccinia virus, vesicular stomatitis virus, and influenza virus.
6 . The oncolytic virus of claim 2 , wherein the oncolytic virus is herpes simplex virus, the nonessential gene is ICP34.5, and the target sequence is inserted into the 5′ or 3′ UTR of one or more essential gene of the oncolytic virus, wherein the one or more essential gene is ICP27.
7 . The recombinant oncolytic virus of claim 2 , wherein the first promoter is a constitutive promoter.
8 . The oncolytic virus of claim 1 , wherein the second promoter is a human tumor-specific promoter.
9 . The oncolytic virus of claim 1 , wherein the enzyme is Dicer, Drosha, or an Argonaute.
10 . The oncolytic virus of claim 1 , wherein the inhibitory component further comprises expanded nucleotide triplet repeats which inhibit the bioactivity of Drosha, or a non-coding RNA which inhibits Dicer activity.
11 . The oncolytic virus of claim 8 , wherein the second expression cassette further contains an enhancer sequence for enhancing expression of the inhibitory component expression sequence.