The present disclosure relates to methods for transiently activating temperature-sensitive agents in one or more cells, for example by contacting one or more cells with a temperature-sensitive agent and transiently incubating the cells at a permissive temperature for inducing an activity of the temperature-sensitive agent in the cells. Additionally, the present disclosure relates to methods of contacting one or more cells in a subject with a temperature-sensitive agent and then lowering the subject's core body temperature to a permissive temperature for inducing an activity of the temperature-sensitive agent in the cells. The disclosure also relates to methods of contacting one or more cells in a subject with a temperature-sensitive agent, maintaining the subject's surface body temperature at a permissive temperature for inducing an activity of the temperature-sensitive agent in the cells. Further disclosed are methods of treating a subject with a temperature-sensitive therapeutic agent.

The present invention relates to a method for producing induced natural killer (iNK) cells, into which a chimeric antigen receptor (CAR) gene encoding a CAR is introduced, iNK cells produced by the method, and a cell therapy composition and a pharmaceutical composition for preventing or treating cancer, comprising the iNK cells.

The method according to the present invention has the effects of producing the iNK cells, into which a CAR gene is introduced, with high efficiency through direct reprogramming from isolated cells without limiting an initial cell, and directly producing the same without a differentiation process, thereby simplifying the production process and reducing costs and time. The method according to the present invention has the effect of producing excellent NK cells having enhanced safety by directly producing NK cells from human somatic cells that are easy to obtain, without passing through induced pluripotent stem cells produced through conventional reprogramming technology. In addition, the iNK cells, into which a CAR gene is introduced, produced by the method, have an excellent cancer cell killing ability, and thus can be effectively utilized as a cell therapy composition or a pharmaceutical composition for preventing or treating cancer.

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.

An objective of the present invention is to provide vectors for conveniently and efficiently producing ES-like cells in which foreign genes are not integrated into the chromosome. The present inventors discovered methods for producing ES-like cells from somatic cells using chromosomally non-integrating viral vectors. Since no foreign gene is integrated into the chromosome of the produced ES-like cells, they are advantageous in tests and research, and immunological rejection and ethical problems can be avoided in disease treatments.

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 provides methods for improving the efficiency of inducing pluripotent stem cells, as well as vectors and compositions for use therein. In the induction of pluripotent stem cells which contains the step of introducing a vector that contains the KLF gene, OCT gene, and SOX gene in this order, the efficiency of pluripotent stem cell induction was successfully increased significantly by further introducing a vector that contains the KLF gene but not the OCT gene and the SOX gene. The methods of the present invention have an excellent feature in that they allow efficient induction of pluripotent stem cells under a temperature condition closer to the physiological environment, and prompt vector removal after the pluripotent stem cell induction. The present invention enables more efficient induction of pluripotent stem cells.

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.

An object of the present invention is to provide a method for producing a reversibly immortalized cell which can allow a cell into which an immortalizing gene is introduced to proliferate over a long period without damaging the chromosome of the cell, and is capable of removing the immortalizing gene, and to provide a method for obtaining a large amount of a reversibly immortalized cell that can be cloned and has stable quality. The present invention provides a method for producing a reversibly immortalized cell, comprising the steps of: introducing a chromosomally non-integrated RNA virus vector loaded with one or two or more immortalizing gene(s), selected from the group consisting of Bmi-1 gene, TERT gene, and SV40T gene, into a mammalian cell so that the immortalizing gene is expressed in the cell; and culturing the obtained cell for proliferation.

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.

The present invention relates to a vector(s) containing and expressing an optimized HIV EnvF gene, methods for making the same and cell substrates qualified for vaccine production which may comprise vector(s) containing optimized HIV genes.