Protein production and protein delivery

A method of targeting DNA sequences into genomic DNA of a cell of vertebrate origin, comprising the steps of:

• a) providing a DNA construct comprising:
• 1) exogenous DNA encoding a product to be expressed in cells of vertebrate origin;
• and
• 2) DNA sequences homologous with genomic DNA sequences in the cell of vertebrate origin;
• and
• 3) amplifiable DNA sequences encoding a marker for which selection of amplified copies of the marker can be performed;
• b) introducing into cells the DNA construct provided in (a), thereby producing cells containing the DNA construct provided in (a);
• c) maintaining cells produced in (b) under conditions appropriate for homologous recombination to occur between DNA sequences homologous with genomic DNA sequences and genomic DNA sequences,
• thereby producing homologously recombinant cells of vertebrate origin having the DNA construct of (a) integrated into genomic DNA of the cells;
• and
• d) culturing the homologously recombinant cells produced in (b) under conditions which select for amplification of the amplifiable DNA encoding a selectable marker, whereby the amplifiable DNA encoding a selectable marker and the endogenous gene functionally linked to exogenous DNA of (a)(1) are coamplified,
• thereby producing homologously recombinant cells containing amplified DNA encoding a selectable marker and a coamplified endogenous gene functionally linked to DNA sequences of (a)(1), wherein the cells are capable of expressing the coamplified endogenous gene.

A method for the in vitro production of a therapeutic protein using a microbial cell containing an intronless copy of a human gene encoding a therapeutic protein, comprising the steps of:

• a) introducing into human cells a DNA construct comprising:
• 1) a retroviral LTR;
• 2) a gene encoding a marker for selection in microbial cells;
• 3) regulatory sequences capable of promoting expression of the therapeutic protein in microbial cells;
• 4) DNA encoding a leader peptide capable of promoting secretion of the therapeutic protein from microbial cells;
• and
• 5) sequences sufficient to direct homologous recombination of the DNA construct with human genomic DNA sequences adjacent to and upstream of DNA encoding the first amino acid in the therapeutic protein,
• thereby producing cells containing the DNA construct;
• b) maintaining the cells produced in (a) under conditions appropriate for homologous recombination to occur between DNA sequences homologous with genomic DNA sequences and genomic DNA sequences, thereby producing homologously recombinant cells having the DNA construct of (a) integrated into genomic DNA adjacent to and upstream of and functionally linked to the gene encoding the therapeutic protein;
• c) introducing into the homologously recombinant cells produced in (b) a DNA construct comprising:
• 1) DNA sequences capable of directing transcription termination in a microbial cell;
• 2) DNA sequences capable of directing DNA replication in a microbial cell;
• 3) a retroviral LTR;
• 4) sequences sufficient to direct homologous recombination of the DNA construct with human genomic DNA sequences downstream of the stop codon of the therapeutic protein, thereby producing homologously recombinant cells having integrated the DNA construct of (a) into the genomic DNA, and containing the DNA construct of (c);
• d) maintaining the cells produced in (c) under conditions appropriate for homologous recombination to occur between DNA sequences homologous with genomic DNA sequences and genomic DNA sequences, thereby producing homologously recombinant cells having the DNA construct of (a) integrated into genomic DNA upstream of and functionally linked to the gene encoding the therapeutic protein, and also having the DNA construct (c) integrated downstream of and functionally linked to the gene encoding the therapeutic protein;
• e) culturing the cells produced in (d) under conditions appropriate for retroviral LTR directed transcription, processing and reverse transcription of the RNA product of the DNA sequences between the LTRs introduced in (a) and (c), thereby producing a DNA sequence comprising an intronless DNA copy of the gene encoding the therapeutic protein operatively linked to the DNA sequences comprising the DNA construct described in (a) and operatively linked to the DNA sequences comprising the DNA construct described in (c), this sequence herein referred to as the intronless DNA sequence;
• f) separating the intronless DNA sequence produced in (e) from the cells;
• g) introducing the DNA separated in (f) into microbial cells, thereby producing microbial cells containing the DNA produced in (e);
• h) culturing the microbial cells produced in (g) in the presence of a selective agent which selects for the selectable marker present in the DNA construct described in (a);
• i) culturing the cells produced in (h) under conditions appropriate for expression and secretion of the therapeutic protein in microbial cells, thereby producing the therapeutic protein in the microbial cells and secreting the protein therefrom;
• and
• j) separating the therapeutic protein produced in (i) from the microbial cells.