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

Abstract

We constructed a hirudin cDNA cassette, HV-1.1, that and mass spectroscopic analysis revealed the presence of encodes mature hirudin variant-1 fused to the signal pepan extra N-terminal serine residue, indicating aberrant tide of human tissue-type plasminogen activator (t-PA).cleavage of the t-PA signal peptide and likely accounting The cassette was subcloned into retroviral vectors and for the diminished activity. We therefore constructed a used to transduce human vascular endothelial cells in vitro.second cDNA cassette, HV-1.2, in which hirudin secretion Hirudin antigen and activity were measured by ELISA and was directed by the signal peptide of human growth horthrombin inhibition assays, respectively. Transduced cells mone. Hirudin expressed from the HV-1.2 cassette had a secreted up to 35 ± 2 ng/10 6 cells/24 h of biologically active specific activity of 13.5 ± 0.2 ATU/ g. Protein sequencing hirudin; expression was stable for at least 7 weeks.and mass spectroscopic analysis demonstrated proper Recombinant hirudin, expressed from the HV-1.1 cassette, cleavage of the growth hormone signal peptide. Thus, we had a specific activity of 7.1 ± 0.2 antithrombin units per achieved high level retrovirus-mediated secretion of biomicrogram (ATU/ g), compared with specific activities of logically active hirudin from endothelial cells in vitro. Use approximately 12 ATU/ g for both native leech hirudin and of these vectors may permit sustained local antagonism of recombinant hirudin produced in yeast. Protein sequencing thrombin activity in vivo.

Expression of recombinant anticoagulant hirudin in the differentiated cultures of the porcine mammary epithelial cell line SI‐PMEC

“…It can be proposed that the band was indeed present, with a molecular mass corresponding to that of hirudin trimers identified by the monoclonal anti-hirudin antibody. This speculation is consistent with the report by Rade et al (1999).…”

“…1A), produced recombinant protein with a molecular mass of 18e20 kDa as determined by Western blotting. In fact, a similar phenomenon was also shown for the commercial recombinant hirudin (Rade et al, 1999). The immunoreactive bands present in the commercial hirudin and the homogenate of SI-PMEC cells did not agree with the predicted 6e7 kDa form of hirudin.…”

Vascular Gene Therapy

“…Examples of such genes include tissue plasminogen activator (t-PA), which activates plasminogen to plasmin that can then mediate fibrinolysis 65,66 ; tissue factor pathway inhibitor, because tissue factor is the primary stimulator of the coagulation pathway 67 ; and hirudin. 68,69 These genes may be very useful in preventing early thrombosis following bypass surgical or angioplasty procedures.…”

Gene therapy for vein graft disease

“…Rade et al [19,20] have utilized both ade-noviral and retroviral vectors to transfer a hirudin gene into vessels. Rade et al [19,20] have utilized both ade-noviral and retroviral vectors to transfer a hirudin gene into vessels.…”