Disclosed is a new process for the production of recombinant proteins, by transient transfection of suspension-grown human embryonic kidney cells (293 cell line and its genetic variants) with an expression vector, using polyethylenimine (PEI) as a transfection reagent. In a preferred embodiment, the process uses 293E cells expressing the Epstein-Barr virus (EBV) EBNA 1 protein, in combination with an oriP-based episomal expression vector having an improved cytomegalovirus expression cassette comprising the CMV5 promoter. The process combines in a single step the cell growth, transfection and protein expression, is carried out without changing the culture medium, and allows to achieve high expression levels in a short period of time. The process may be carried out in a serum-free, low-protein culture medium, is easily scalable, compatible with continuous production processes, and fully adapted to high-throughput production of milligram quantities of recombinant proteins.

The invention provides cells and animals, as well as methods of producing cells and animals, that express at least one interfering RNA molecule to regulate the expression of a specific gene or family of genes. The invention further provides novel iRNA molecules, as well as DNA templates for producing iRNA molecules.

The present invention relates to an expression cassette for a target protein, comprising a promoter, a polynucleotide coding for the target protein, an intron sequence, and a poly A sequence, an expression vector, and a transformant. The expression cassette for a target protein according to the present invention can simultaneously perform the expression of the intron sequence and the target protein through one transduction and exhibits the effect of inducing the high expression and high functionality of the target protein by regulating the expression of an endogenous gene.

An adenovirus comprising a sequence of formula (I) 5ITR-B.sub.1-B.sub.A-B.sub.2-B.sub.X-B.sub.B-B.sub.Y-B.sub.3-3ITR wherein By comprises a transgene cassette containing four transgenes, said genes encoding a FAP-BITE, CXL10, CXL9, and IFN. The disclosure also extends to a pharmaceutical composition comprising the virus, and use of the virus or formulation in treatment.

Methods and compositions for modifying the coding sequence of endogenous genes using rare-cutting endonucleases and transposases. The methods and compositions described herein can be used to modify the coding sequence of endogenous genes.

Two or more conditional, dominant, lethal gene expression systems provide high levels of penetrance in insects. Lethality is induced at an earlier stage of development and the risk of biochemical resistance is reduced, as compared to a single insect conditional, dominant, lethal gene expression system. The invention is useful for the control of insect populations.

Methods and compositions for modifying the coding sequence of endogenous genes using rare-cutting endonucleases and transposases. The methods and compositions described herein can be used to modify the coding sequence of endogenous genes.

The invention provides a platform and methods of using the platform for the regulation of the expression of a target gene using exposure to an aptamer ligand (for example, a small molecule). The platform features a polynucleotide gene regulation cassette that is placed in the target gene and includes a synthetic riboswitch positioned in the context of a 5 intron-alternative exon-3 intron. The riboswitch comprises an effector region and a sensor region (e.g., an aptamer that binds a small molecule ligand) such that the alternative exon is spliced into the target gene mRNA when the ligand is not present thereby preventing expression of the target gene. When the ligand is present, the alternative exon is not spliced into the target gene mRNA thereby providing expression of the target gene.

Disclosed are methods and constructs for engineering circular RNA. Disclosed is a vector for making circular RNA, said vector comprising the following elements operably connected to each other and arranged in the following sequence: a.) a 5 homology arm, b.) a 3 group I intron fragment containing a 3 splice site dinucleotide, c.) optionally, a 5 spacer sequence, d.) a protein coding or noncoding region, e.) optionally, a 3 spacer sequence, f) a 5 Group I intron fragment containing a 5 splice site dinucleotide, and g.) a 3 homology arm, said vector allowing production of a circular RNA that is translatable or biologically active inside eukaryotic cells. In another embodiment, the vector can comprise the 5 spacer sequence, but not the 3 spacer sequence. In yet another embodiment, the vector can comprise the 3 spacer sequence, but not the 5 spacer sequence. Also disclosed is a method for purifying the circular RNA produced by the vector and the use of nucleoside modifications in circular RNA produced by the vector.

The invention provides a method for generating a transgenic eukaryotic cell population having a modified human Rosa26 locus, which method includes introducing a functional DNA sequence into the human Rosa26 locus of starting eukaryotic cells. Also provided are targeting vectors useful in the method, as well as a cell population and a transgenic non-human animal comprising a modified human Rosa26 locus. Finally, the invention provides an isolated DNA sequence corresponding to the human Rosa26 locus.