Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins

We constructed a hirudin cDNA cassette, HV-1.1, that encodes mature hirudin variant-1 fused to the signal peptide of human tissue-type plasminogen act ivator (t-PA). The cassette was subclonced into retroviral vectors and used to transduce human vascular endothelial cells in vitro. Hirudin antigen an d activity were measured by ELISA and thrombin inhibition assays, respectiv ely. Transduced cells secreted up to 35 +/- 2 ng/10(6) cells/24 h of biolog ically active hirudin; expression was stable for at least 7 weeks. Recombin ant hirudin, expressed from the HV-1.1 cassette had a specific activity of 7.1 +/- 0.2 antithrombin units per microgram (ATU/mu g), compared with spec ific activities of approximately 12 ATU/mu g for both native leech hirudin and recombinant hirudin produced in yeast. Protein sequencing and mass spec troscopic analysis revealed the presence of an extra N-terminal serine resi due, indicating aberrant cleavage of the t-PA signal peptide and likely acc ounting for the diminished activity We therefore constructed a second cDNA cassette, HV-1.2, in which hirudin secretion was directed by the signal pep tide of human growth hormone. Hirudin expressed from the HV-1.2 cassette ha d a specific activity of 13.5 +/- 0-2 ATU/mu g. Protein sequencing and mass spectroscopic analysis demonstrated proper cleavage of the growth hormone signal peptide. Thus, we achieved high level retrovirus-mediated. secretion of biologically active hirudin from endothelial cells in vitro. Use of the se vectors may permit sustained local antagonism of thrombin activity in vi vo.