A nucleic acid constructs are used in improving site-specific insertion of an exogenous nucleic acid into a genome. In some aspects the nucleic acid construct having a first polynucleotide sequence encoding a DNA binding protein engineered to bind to a specific genomic DNA sequence, a second polynucleotide having a modified integrase or a modified transposase that enables insertion of exogenous nucleic acid into the genome, and a nucleic acid sequence encoding a linker between the two nucleotides. In some aspects, the nucleic acid construct encodes a fusion protein, for example, a fusion protein for delivery to a cell by a lentiviral particle.

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 presently disclosed subject matter relates to targeted integration (TI) host cells suitable for the expression of recombinant proteins wherein those TI host cells have been subjected to supertransfection resulting in the random integration (RI) of exogenous nucleic acids encodes into their genome, as well as methods of producing and using said supertransfected TI host cells.

The invention can be used in biotechnology, in particular to the gene therapy DNA vector GDTT1.8NAS12, Escherichia coli strain JM 110-NAS, Escherichia coli strain JM 110-NAS/GDTT1.8NAS12, and the industrial production of the gene therapy DNA vector GDTT1.8NAS12. A method of obtaining the gene therapeutic DNA vector GDTT1.8NAS12 involves construction of a 2408-bp intermediate vector containing a 688-bp replication origin, a 467-bp transcription terminator hGH-TA, a 137-bp regulatory site RNA-out transposon TnIO, a I0I8-bp kanamycin resistance gene, and a 68-bp polylinker. Then, it is splitted using restriction endonucleases SalI and BamHI and ligated to the promoter regulatory region containing the 1219-bp promoter region of the human elongation factor EF1A with its own enhancer. The kanamycin resistance gene is cleaved at the SpeI restriction sites.

An object of the present invention is to improve the efficiency of a method for producing chimeric antigen receptor (CAR)-expressing cells. The present invention provides a method for producing genetically modified mammalian cells, comprising the steps of: a) introducing a polynucleotide encoding a chimeric antigen receptor (CAR) protein to a cell population comprising T cells derived from a mammal by a transposon method to obtain a genetically modified cell population; b) providing an endogenous cell population derived from the mammal expressing a protein that binds to the CAR; and c) coculturing the genetically modified cell population of the step a) and the endogenous cell population of the step b).

The present invention provides nucleic acids, vectors, host cells, methods and compositions to confer and/or augment immune responses mediated by cellular immunotherapy, such as by adoptively transferring CD8+ central memory T cells or combinations of central memory T cells with CD4+ T cells that are genetically modified to express a chimeric receptor. In some alternatives the genetically modified host cell comprises a nucleic acid comprising a polynucleotide coding for a ligand binding domain, a polynucleotide comprising a customized spacer region, a polynucleotide comprising a transmembrane domain, and a polynucleotide comprising an intracellular signaling domain. In some alternatives, the ligand binding domains binds to CD171.

The present invention relates to processes for transfecting cells. In particular, the present invention relates to processes for using CRISPR to incorporate a polynucleotide into the genome of an avian primordial germ cell (PGC).

The present invention relates to methods for administering autologous and/or allogeneic B cells genetically modified to produce a therapeutic agent, such as a therapeutic protein. Specifically disclosed are methods for administering a single, maximally effective dose of genetically modified B cells and for administering multiple doses of genetically modified B cells. The compositions and methods disclosed herein are useful for the long-term, in vivo delivery of a therapeutic agent.

Methods and composition for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed.

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. Some of the vectors are useful for tetracycline-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 a method of producing a protein of interest, in vitro, involving the mammalian host cell.