An objective of the present invention is to provide an improved negative-strand RNA viral vector and a use thereof, the negative-strand RNA viral vector exhibiting transient high expression of genes loaded in the vector and enabling the rapid removal of the vector after said expression. It was discovered that by adding a micro-RNA target sequence to the NP, P, or L gene of a negative-strand RNA viral vector, it is possible to control the expression of the vector depending on the micro-RNA expressed by the introduction cell. In particular, when a micro-RNA target sequence was added to the NP or P gene, the expression of the vector decreased depending on the micro-RNA, and the removal of the vector was promoted, while the effect was reversed when a micro-RNA target sequence was added to the L gene. The vector can be applied in cell therapy and regenerative medicine and can be used as a therapeutic vector that targets cancer.

The present invention provides novel donor polynucleotides formed by linking the two ends of a genomic fragment containing a cleavable site by a polynucleotide carrying a positive selection marker gene and a negative selection marker gene. Use of the donor polynucleotide makes it possible to modify only a target gene with avoiding the possibility of introducing mutations to sequences, called off-target, which are other than the target sequence, by introducing cleavage in a homologous site of the donor polynucleotide without introducing cleavage in a target gene locus.

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.

In this invention, a non-infectious particle has been produced, comprising a pathogen antigen protein caused to be expressed on the surface of a virus particle having at least one species of paramyxovirus envelope protein missing from the particle. This particle has been found to hold within the particle a large amount of antigen protein compared to an infectious particle, and to be capable of eliciting a host immune response with extremely high efficiency. The non-infectious particle according to the present invention is useful as a vaccine against a pathogenic virus, or the like.

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.

Methods of gene correction, methods of generating induced pluripotent stem cells (iPSCs), and methods of deriving multi-lineage cell types with therapeutic value. In some embodiments, the gene correction affects the expression and/or function of the functional type VII collagen protein (C7).

The present disclosure relates to viral delivery of RNA utilizing self-cleaving ribozymes and applications of such, including but not limited to CRISPR-Cas related applications.

Simultaneous 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; (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; (7) an RNA sequence that codes for a protein for regulating the activity of the polymerase; and (8) an RNA sequence that codes for the single-strand RNA binding protein.

Provided is a method of generating iPS cells. Specifically, provided is a method of generating iPS cells having a rearranged -TCR gene. Also provided is a cell population including the generated iPS cells. The method includes stimulating collected blood cells with IL-2 and a bisphosphonate, and then introducing cell reprogramming factors through use of a Sendai virus (SeV) vector. According to the method of the present invention, iPS cells having a rearranged -TCR gene can be effectively generated. In particular, the method may be free of a step of treating the blood cells with an antibody before the step of stimulating blood cells with any one kind or a plurality of kinds of interleukins selected from IL-2, IL-15, and IL-23, and a bisphosphonate. In addition, iPS cells generated by the method of the present invention can be differentiated into desired cells by differentiation induction treatment.

The present invention relates to retroviral vectors, particularly lentiviral vectors, comprising a modified retroviral RNA sequence that is codon-substituted and comprises a reduced number of retroviral open-reading frames, and wherein the retroviral vector is pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, methods of making the same and uses thereof.