Gene expression system using stealthy RNA, and gene introduction/expression vector including said RNA

Inventors

Nakanishi, Mahito • Iijima, Minoru

Assignees

Tokiwa-Bio Inc • National Institute of Advanced Industrial Science and Technology AIST

Abstract

The present invention enables simultaneous and stable expression of a plurality of foreign genes by using a stealthy RNA gene expression system that is a complex that does not activate the innate immune mechanism and is formed from an RNA-dependent RNA polymerase, a single-strand RNA binding protein, and negative-sense single-strand RNAs including the following (1) to (8): (1) a target RNA sequence that codes for any protein or functional RNA; (2) an RNA sequence forming a noncoding region and derived from mRNA expressed in animal cells; (3) a transcription initiation signal sequence recognized by the RNA-dependent RNA polymerase; (4) a transcription termination signal sequence recognized by the polymerase; (5) an RNA sequence containing a replication origin recognized by the polymerase; (6) an RNA sequence that codes for the polymerase and of which codons are optimized for the species from which an introduction target cell is derived; (7) an RNA sequence that codes for a protein for regulating the activity of the polymerase and of which codons are optimized for the species from which the introduction target cell is derived; and (8) an RNA sequence that codes for the single-strand RNA binding protein and of which codons are optimized for the species from which the introduction target cell is derived.

Core Innovation

The invention provides a stealth RNA gene expression system comprising a negative-sense single-stranded RNA (A), a single-stranded RNA binding protein (B), and an RNA-dependent RNA polymerase (C). The stealth RNA gene expression system is a complex that does not activate an innate immune system. The negative-sense single-stranded RNA (A) includes RNA sequences encoding any given protein or functional RNA, together with noncoding region(s), transcription start signal sequences, transcription termination signal sequences, and RNA sequences comprising replication origins.

The noncoding region(s) in the negative-sense single-stranded RNA include partial mRNA(s) of an animal gene expressed in animal cells, and the transcription start signal sequences, transcription termination signal sequences, and replication origins are isolated from a genome of the same virus as the RNA virus. The RNA sequences encoding the RNA-dependent RNA polymerase, the protein that regulates activity of the RNA-dependent RNA polymerase, and the single-stranded RNA binding protein are specific to L protein, C protein, and NP protein of an RNA virus belonging to a paramyxovirus family.

The invention also includes a stealth RNA that is the negative-sense single-stranded RNA (A) itself, structured to be unrecognizable by an innate immune system. This stealth RNA includes noncoding region(s) that are unrecognizable by an innate immune system, and sequences for transcription start, transcription termination, and replication origins recognized by the RNA-dependent RNA polymerase, so that forming the complex with the RNA binding protein (B) and the RNA-dependent RNA polymerase (C) does not activate innate immune system.

Claims Coverage

Two independent claims are provided, covering the stealth RNA gene expression system complex and the stealth negative-sense single-stranded RNA itself. The claims specify four inventive features across the independent claim set.

Across the independent claims, the core inventive coverage is directed to a stealth negative-sense single-stranded RNA complex and to a stealth RNA definition with innately unrecognizable noncoding regions plus defined transcription and replication elements, together enabling complex formation that does not activate an innate immune system.

Stated Advantages

Documented Applications