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.

Disclosed herein are compositions and methods for treating cancer in a subject. This involves administering an oncolytic virus containing a heterologous DNA sequence encoding one or more immunomodulatory and/or immunostimulatory polypeptide(s) of interest to the subject under conditions effective to enhance an anti-tumor immune response in the subject, and to treat cancer. It also relates to a method of enhancing the delivery to and distribution within a tumor mass of therapeutic viruses.

The present invention relates to expression vectors for the heterologous expression of a nucleic acid sequence of interest in mammalian cells, the vectors comprising a chimeric promoter regulatory sequence being operably linked to a nucleic acid sequence to be expressed, wherein the chimeric promoter regulatory sequence comprises a cytomegalovirus promoter sequence derived from murine cytomegalovirus or from human cytomegalovirus and being operably linked to the transcriptional start site of the nucleic acid sequence to be expressed; and a cytomegalovirus upstream region and/or enhancer sequence derived from human and/or the simian cytomegalovirus, wherein the upstream region and/or enhancer sequence is located 5 of and operably linked to the murine or the human promoter sequence, and wherein the chimeric promoter regulatory sequence comprises sequence elements being derived from at least two of the group consisting of murine cytomegalovirus, human cytomegalovirus and simian cytomegalovirus. In particular embodiments, the chimeric promoter regulatory sequence comprises sequence elements derived from the murine or the human cytomegalovirus IE1 promoter and from the human and/or the simian cytomegalovirus IE1 region. The invention also relates to mammalian host cells transfected with such expression vectors, a method for heterologous expression of a nucleic acid sequence in a mammalian host cell by employing such expression vectors, and the use of such expression vectors for the heterologous expression of a nucleic acid sequence.

Recombinant expression vectors are disclosed that include a control sequence for recombinant expression of proteins of interest; the control sequence combines a mCMV enhancer sequence with a rat EF-1alpha intron sequence or a human cytomegalovirus (huCMV) intron A sequence. Some of the vectors are useful for inducible expression. Some of the vectors contain a 5 PiggyBac ITR and a 3 PiggyBac ITR to promote genomic integration into a host cell chromosome. A method of selecting a stable production cell line for manufacturing a protein of interest is also disclosed. Also disclosed are mammalian host cells comprising the inventive recombinant expression vectors and methods of producing a protein of interest, in vitro, involving the mammalian host cell.

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.

Disclosed herein are recombinant baculoviruses suitable for introducing an exogenous gene into a pest insect, particularly, disease-transmitting mosquitos. The recombinant baculovirus is characterized in having a promotor that is any of a HzNV-1 viral early expressing gene pag1, a ceropin gene b1, a defensin gene a4, or hp70 gene; and an exogenous gene operably linked thereto the promoter. Also disclosed herein is a method of introducing an exogenous gene into a pest insect. The method includes transducing the pest insect with a recombinant baculovirus without suppressing the production of microRNAs (miRNAs) in the pest insect, wherein the recombinant baculovirus comprises a promoter of pag1, cecropin b1, defensin gene a, or hp70.

The current invention reports a promoter that has the nucleic acid sequence of SEQ ID NO: 02 or SEQ ID NO: 03 which is a human CMV major immediate-early (hCMV-MIE) promoter/enhancer with C to G point mutation at position 41 and/or 179 relative to the transcription start site. This new promoter is especially useful for the production of polypeptides at large scale as it shows reduced promoter silencing and improved polypeptide production.

The invention features compositions and methods for identifying orthogonal transcriptional switches derived from Tet repressor homologs for Saccharomyces cerevishiae regulated by 2,4-diacetylphloroglucinol (DAPG) and other ligands.

Compositions and methods for the detection of rare target polynucleotides in a complex mixture.

A strong insulator fragment from foamy virus, which can be used to insulate expression of a transgene and reduce genotoxicity of integrating vectors comprising such. The insulator fragment can also be used in gene targeting constructs in gene editing.