Retroviral Vector-Mediated Expression of Hirudin by Human

Gene Therapy (1999) 6, 385–392 1999 Stockton Press All rights reserved 0969-7128/99 $12.00 http://www.stockton-press.co.uk/gt Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins JJ Rade1,2, M Cheung1, S Miyamoto1 and DA Dichek1,3 1Molecular Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, MD; 2Division of Cardiology, Johns Hopkins School of Medicine, Baltimore, MD; and 3Gladstone Institute of Cardiovascular Disease and Department of Medicine, University of California San Francisco, CA, USA 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 pep- an 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 hor- thrombin inhibition assays, respectively. Transduced cells mone. Hirudin expressed from the HV-1.2 cassette had a secreted up to 35 ± 2 ng/106 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 bio- microgram (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. Keywords: gene therapy; endothelial cells; hirudin; signal peptide; thrombin Introduction med in several animal model systems, have demonstrated that systemically administered hirudin can Autologous venous and prosthetic grafts are widely used prevent thrombosis and neointimal proliferation after as conduits for peripheral and coronary artery bypass arterial injury.7–10 Systemically administered hirudin has surgery. The utility of these conduits is limited, however, also been used in humans as an adjunct to thrombolytic by thrombosis and neointimal hyperplasia, both of which therapy and coronary angioplasty. Although some short- may result from generation of thrombin on the luminal term benefits of hirudin infusion have been reported, sys- graft surface. Thrombin activates platelets and cleaves temic hirudin administration has failed to prevent resten- 1 fibrinogen, leading to thrombosis and vessel occlusion. osis and has been associated in some settings with unac- Thrombin can also contribute to the development of graft ceptable bleeding complications.11–14 The hemorrhagic neointimal hyperplasia through its action as a smooth complications associated with systemic hirudin adminis- muscle cell mitogen, a mediator of inflammation, and tration may preclude the use of doses that are sufficient an agonist for the release of platelet-derived growth to achieve local thrombin inhibition at sites of vascular 2–5 factor. disease. Hirudin, a 65-amino acid protein secreted by the sali- We have previously demonstrated that adenovirus- vary glands of the medicinal leech, is a potent and spe- mediated gene transfer of hirudin in injured rat arteries 6 cific inhibitor of thrombin. By blocking both the catalytic results in hirudin secretion from the injured artery at a site and the anion-binding exosite of thrombin, hirudin level that is adequate to reduce neointimal formation.15 inhibits the interaction of thrombin both with fibrinogen Importantly, this effect was achieved without evidence of and with thrombin receptors that are present on the sur- systemic anticoagulation. Although adenovirus-mediated face of platelets and vascular cells. Recent studies, perfor- delivery of hirudin or other potentially therapeutic proteins to the vessel wall is relatively efficient, adenovirus- encoded transgene expression is transient and may be Correspondence: JJ Rade, Division of Cardiology, Johns Hopkins School of Medicine, Carnegie 565-C, 600 N Wolfe St, Baltimore, MD 21287- associated with a profound local inflammatory 16 6568, USA response. In addition, the high prevalence of pre-exist- Received 29 June 1998; accepted 24 September 1998 ing immunity to adenovirus in the human population Hirudin expression from transduced endothelium JJ Rade et al 386 may limit the ability of adenoviral vectors to deliver genetic material in a clinical setting.17 Retroviral vectors offer a promising alternative to adenoviral vectors for vascular gene therapy. Because retroviral vectors can insert genetic material into target cell genomic DNA, they have the potential to achieve stable long-term transgene expression. Moreover, many retroviral vectors are devoid of open reading frames encoding viral proteins. Elimination of these open reading frames should prevent destructive host inflammatory responses that are directed against foreign proteins expressed from transduced cells.18 Although achievement of efficient in vivo gene transfer to blood vessels has been a major problem with retroviral vectors,19,20 a recent report suggests that significant levels of in vivo retrovirus-mediated gene transfer may now be feasible.21 Alternatively, vascular endothelial cells transduced ex vivo with retroviral vectors might be introduced into the vasculature by seeding of these cells on to autologous vascular grafts or synthetic prostheses. We and others have demonstrated the feasibility of seeding retrovirally transduced endothelial cells on to prosthetic grafts, endo- vascular metal stents and native denuded arteries.22–24 As initial steps toward achievement of sustained local Figure 1 Retroviral vectors and cassettes expressing recombinant hirudin. (a) G HV-1.1 and G SHV-1.1 retroviral vectors containing the HV-1.1 thrombin inhibition via retroviral transduction of vascu- 1 1 cassette and G1HV-1.2 vector containing the HV-1.2 cassette. The vectors lar cells, we constructed retroviral vectors expressing consist of the Moloney murine leukemia virus 5Ј and 3Ј long terminal hirudin. We used vector backbones with and without repeats (LTR), non-coding regions (thin lines) containing the packaging internal splice donor and acceptor sites and constructed signal (⌿+), the HV-1.1 or HV-1.2 cassettes, the simian virus-40 early hirudin cDNA expression cassettes using two different promoter (SV40) and the neomycin phosphotransferase gene (NeoR) which heterologous signal peptide sequences to direct hirudin confers resistance to the neomycin analog G418. G1SHV-1.1 contains splice donor (SD) and splice acceptor (SA) sites, allowing synthesis of a secretion. Finally, the vectors were tested for their ability spliced mRNA encoding HV-1.1. (b) Hirudin cDNA cassettes, HV-1.1 to direct the secretion of intact, biologically active hirudin and HV-1.2, containing the coding sequence for mature hirudin variant- from cultured human endothelial cells. 1 (HV-1) downstream of the coding sequence for either the human t-PA signal peptide (t-PA Pre) or the human growth hormone signal peptide (hGH Pre). Also shown are the 5Ј and 3Ј untranslated sequences (thick Results black lines) and flanking sequences in the cloning vector (thin black lines) containing the restriction sites used for subcloning. Quantification of hirudin secretion from endothelial cells transduced with vectors expressing HV-1.1 Hirudin antigen and thrombin inhibitory activity were Table 1 Retrovirus-mediated hirudin expression by transduced measured in 72 h collections of conditioned medium har- endothelial cells vested from untransduced human umbilical vein endo- 6 6 thelial cells (HUVEC) as well as from HUVEC transduced Vector ng/10 cells/24 h ATU/10 cells/24 h with the retroviral vectors G1HV-1.1, G1SHV-1.1 (Figure = ± ± G1HV-1.1 (n 6) 35 2 0.07 0.01* 1a), or the control vector G1XSVNa (not shown). = ± ± G1SHV-1.1 (n 6) 19 1 0.04 0.01 G1XSVNa lacks a hirudin expression cassette but is other- = ± ± G1HV-1.2 (n 4) 25 1 0.46 0.08* = ± wise identical to G1HV-1.1. Both the G1HV-1.1 and G1XSVNa (n 6) ND 0.03 0.01 = ± G1SHV-1.1 vectors contain the HV-1.1 hirudin expression None (n 6) ND 0.02 0.01 cassette, in which a synthetic cDNA for mature hirudin variant 1 is fused to the coding sequence for a human ATU indicates antithrombin units; ND, not detectable. Values tissue plasminogen activator (t-PA) signal peptide are expressed as mean ± s.e.m. Ͼ ± *P 0.02 compared with G1XSVNa. (Figure 1b). G1HV-1.1-transduced cells secreted 35 2 ng/106 cells/24 h of hirudin antigen, which was associated with a low level of thrombin inhibitory activity (Table 1). Secretion of hirudin antigen from G1SHV-1.1-trans- were analyzed by Western blotting (Figure 2). Native duced HUVEC was somewhat lower, demonstrating that hirudin can exist as oligomers at neutral pH;25 therefore, the existence of splice donor and acceptor sites in this we anticipated that several species would be identified construct did not increase protein expression levels. by the polyclonal anti-hirudin antibody. Several bands Thrombin inhibitory activity in the supernatant of were indeed present, with molecular masses correspond- G1SHV-1.1-transduced cells did not differ significantly ing to those of hirudin trimers, dimers and monomers. from controls. The pattern of bands detected in the lanes containing recHV-1.1 purified from the conditioned media of retro- Western blot analysis virus-transduced cells was similar to that present in lanes Recombinant hirudin (recHV-1.1) samples purified from containing either native leech hirudin or recombinant the conditioned medium of G1SHV-1.1-transduced cells hirudin produced in yeast.

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