Various embodiments are disclosed herein relate to methods for selection of a genetically engineered cell. Some embodiments relate to a cell that is produced with the methods disclosed herein.

The present disclosure reports that a calcium-dependent serine protease, complement component is (C1s) has been identified as a protease responsive for cleavage of exogenous polypeptides expressed in mammalian cell lines such as CHO cells. These CHO cell lines provide for increased yield of antigenically correct, uncleaved exogenous polypeptide as compared to unmodified CHO cells expressing the active C1s protease. C1s-deficient cell lines and methods for use of same for producing exogenous polypeptides, e.g., human immunodeficiency virus (HIV) envelope glycoprotein polypeptides, such as, gp120 or human Factor VIII are provided.

The present disclosure relates to providing an engineered CHO cell line wherein the two essential metabolic genes are knocked out. Particularly, the present invention relates to a double knockout CHO cell line (DHFR−/− and GS−/−) with disrupted Dihydrofolate Reductase (DHFR) and Glutamine Synthetase (GS) genomic loci. The double knockout CHO cell line (DHFR−/− and GS−/−) being suitable for expression of monoclonal antibodies, dimeric therapeutic proteins, Fab, single chain fragments, or the like. The present disclosure also provides method of generation of a double knock out CHO cell line (DHFR−/− and GS−/−) using gene selection and/or manipulating techniques such as CRISPR/Cas9 system, Zinc Finger Nuclease, TALEN, or the like. The present disclosure further provides method of selection of clones and production of therapeutic proteins of interest with increased titre.

The present invention provides mammalian cell expression vectors that impart to mammalian host cells an ability to produce high levels of foreign gene-derived proteins. A ubiquitously acting chromatin opening element (UCOE) is introduced into an expression vector that has a plasmid DNA integrated into a transcriptional hot spot on the chromosome of a dihydrofolate reductase gene-deficient host cell so that it allows for selection of strains that grow in hypoxanthine-thymidine (hereinafter denoted as HT)-free medium, whereby transformants will produce a protein of interest in increased amounts.

Some embodiments of the systems, methods and compositions provided herein relate to a compound protease. In some embodiments, the compound protease includes a protease domain and a cut site for another enzyme. In some embodiments, the compound protease includes an association domain. In some embodiments, the compound protease is part of a protein circuit.

The present invention provides polynucleotide vectors for high expression of heterologous genes. Some vectors further comprise novel transposons and transposases that further improve expression. Further disclosed are vectors that can be used in a gene transfer system for stably introducing nucleic acids into the DNA of a cell. The gene transfer systems can be used in methods, for example, gene expression, bioprocessing, gene therapy, insertional mutagenesis, or gene discovery

The disclosure includes non-naturally occurring or engineered CRISPR Cas9, each associated with at least one destabilization domain (DD), along with compositions, systems and complexes involving the DD-CRISPR Cas9, nucleic acid molecules and vectors encoding the same, delivery systems involving the same, uses therefor.

Disclosed are composition comprising (a) an inducible transgene construct, comprising a sequence encoding an inducible promoter and a sequence encoding a transgene, and (b) a receptor construct, comprising a sequence encoding a constitutive promoter and a sequence encoding an exogenous receptor, wherein, upon integration of the construct of (a) and the construct of (b) into a genomic sequence of a cell, the exogenous reporter is expressed, and wherein the exogenous reporter, upon binding a ligand, transduces an intracellular signal that targets the inducible promoter of (a) to modify gene expression. Methods for introducing compositions into cells and the use of the resultant cells in adoptive cell therapies are also provided.

Some embodiments of the methods and compositions provided herein relate to the preparation surfactant protein-D (SP-D). Some embodiments include the expression of human SP-D in certain cell lines, and the purification of human SP-D from such cell lines. Some embodiments include the preparation of certain oligomeric forms of human SP-D.

The present disclosure is directed to methods and systems for identifying different parts of enzyme structure that can be engineered and/or assisted by engineered technologies to improve the speed and efficiency of the catalyzed chemical reactions. More specifically, the present disclosure is directed to identifying and modifying distal surface regions that affect catalytic activity. These surface regions are identified and ranked for the impact on enzyme activity, based on the transfer of energy from the surface regions to the active site. Methods are described for improving the catalytic efficiency by improving the energy transfer to overcome the activation energy barrier. The methods described are also applicable for improving the stability of enzymes and development of appropriate enzyme immobilization protocols.