Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins
Jj. Rade et al., Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins, GENE THER, 6(3), 1999, pp. 385-392
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.