Retroviral vector-mediated expression of hirudin by human vascular endothelial cells

Implications for the design of retroviral vectors expressing biologically active proteins

J. J. Rade, M. Cheung, S. Miyamoto, D. A. Dichek

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

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 activator (t-PA). The cassette was subcloned into retroviral vectors and used to transduce human vascular endothelial cells in vitro. Hirudin antigen and activity were measured by ELISA and thrombin inhibition assays, respectively. Transduced cells secreted up to 35 ± 2 ng/106 cells/24 h of biologically active hirudin; expression was stable for at least 7 weeks. Recombinant hirudin, expressed from the HV-1.1 cassette, had a specific activity of 7.1 ± 0.2 antithrombin units per microgram (ATU/μg), compared with specific activities of approximately 12 ATU/μg for both native leech hirudin and recombinant hirudin produced in yeast. Protein sequencing and mass spectroscopic analysis revealed the presence of an extra N-terminal serine residue, indicating aberrant cleavage of the t-PA signal peptide and likely accounting for the diminished activity. We therefore constructed a second cDNA cassette, HV-1.2, in which hirudin secretion was directed by the signal peptide of human growth hormone. Hirudin expressed from the HV-1.2 cassette had a specific activity of 13.5 ± 0.2 ATU/μg. 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 these vectors may permit sustained local antagonism of thrombin activity in vivo.

Original languageEnglish (US)
Pages (from-to)385-392
Number of pages8
JournalGene Therapy
Volume6
Issue number3
DOIs
StatePublished - Mar 1999
Externally publishedYes

Fingerprint

Hirudins
Endothelial Cells
Protein Sorting Signals
Proteins
Protein Sequence Analysis
Thrombin
Complementary DNA
Leeches
Antithrombins
Human Growth Hormone
Plasminogen Activators
Tissue Plasminogen Activator
Retroviridae
Serine
Growth Hormone
Yeasts
Enzyme-Linked Immunosorbent Assay

Keywords

  • Endothelial cells
  • Gene therapy
  • Hirudin
  • Signal peptide
  • Thrombin

ASJC Scopus subject areas

  • Genetics

Cite this

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells : Implications for the design of retroviral vectors expressing biologically active proteins. / Rade, J. J.; Cheung, M.; Miyamoto, S.; Dichek, D. A.

In: Gene Therapy, Vol. 6, No. 3, 03.1999, p. 385-392.

Research output: Contribution to journalArticle

@article{0f6e0191cb514b4ba204c15eff7ca39b,
title = "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 encodes mature hirudin variant-1 fused to the signal peptide of human tissue-type plasminogen activator (t-PA). The cassette was subcloned into retroviral vectors and used to transduce human vascular endothelial cells in vitro. Hirudin antigen and activity were measured by ELISA and thrombin inhibition assays, respectively. Transduced cells secreted up to 35 ± 2 ng/106 cells/24 h of biologically active hirudin; expression was stable for at least 7 weeks. Recombinant hirudin, expressed from the HV-1.1 cassette, had a specific activity of 7.1 ± 0.2 antithrombin units per microgram (ATU/μg), compared with specific activities of approximately 12 ATU/μg for both native leech hirudin and recombinant hirudin produced in yeast. Protein sequencing and mass spectroscopic analysis revealed the presence of an extra N-terminal serine residue, indicating aberrant cleavage of the t-PA signal peptide and likely accounting for the diminished activity. We therefore constructed a second cDNA cassette, HV-1.2, in which hirudin secretion was directed by the signal peptide of human growth hormone. Hirudin expressed from the HV-1.2 cassette had a specific activity of 13.5 ± 0.2 ATU/μg. 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 these vectors may permit sustained local antagonism of thrombin activity in vivo.",
keywords = "Endothelial cells, Gene therapy, Hirudin, Signal peptide, Thrombin",
author = "Rade, {J. J.} and M. Cheung and S. Miyamoto and Dichek, {D. A.}",
year = "1999",
month = "3",
doi = "10.1038/sj.gt.3300824",
language = "English (US)",
volume = "6",
pages = "385--392",
journal = "Gene Therapy",
issn = "0969-7128",
publisher = "Nature Publishing Group",
number = "3",

}

TY - JOUR

T1 - Retroviral vector-mediated expression of hirudin by human vascular endothelial cells

T2 - Implications for the design of retroviral vectors expressing biologically active proteins

AU - Rade, J. J.

AU - Cheung, M.

AU - Miyamoto, S.

AU - Dichek, D. A.

PY - 1999/3

Y1 - 1999/3

N2 - 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 activator (t-PA). The cassette was subcloned into retroviral vectors and used to transduce human vascular endothelial cells in vitro. Hirudin antigen and activity were measured by ELISA and thrombin inhibition assays, respectively. Transduced cells secreted up to 35 ± 2 ng/106 cells/24 h of biologically active hirudin; expression was stable for at least 7 weeks. Recombinant hirudin, expressed from the HV-1.1 cassette, had a specific activity of 7.1 ± 0.2 antithrombin units per microgram (ATU/μg), compared with specific activities of approximately 12 ATU/μg for both native leech hirudin and recombinant hirudin produced in yeast. Protein sequencing and mass spectroscopic analysis revealed the presence of an extra N-terminal serine residue, indicating aberrant cleavage of the t-PA signal peptide and likely accounting for the diminished activity. We therefore constructed a second cDNA cassette, HV-1.2, in which hirudin secretion was directed by the signal peptide of human growth hormone. Hirudin expressed from the HV-1.2 cassette had a specific activity of 13.5 ± 0.2 ATU/μg. 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 these vectors may permit sustained local antagonism of thrombin activity in vivo.

AB - 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 activator (t-PA). The cassette was subcloned into retroviral vectors and used to transduce human vascular endothelial cells in vitro. Hirudin antigen and activity were measured by ELISA and thrombin inhibition assays, respectively. Transduced cells secreted up to 35 ± 2 ng/106 cells/24 h of biologically active hirudin; expression was stable for at least 7 weeks. Recombinant hirudin, expressed from the HV-1.1 cassette, had a specific activity of 7.1 ± 0.2 antithrombin units per microgram (ATU/μg), compared with specific activities of approximately 12 ATU/μg for both native leech hirudin and recombinant hirudin produced in yeast. Protein sequencing and mass spectroscopic analysis revealed the presence of an extra N-terminal serine residue, indicating aberrant cleavage of the t-PA signal peptide and likely accounting for the diminished activity. We therefore constructed a second cDNA cassette, HV-1.2, in which hirudin secretion was directed by the signal peptide of human growth hormone. Hirudin expressed from the HV-1.2 cassette had a specific activity of 13.5 ± 0.2 ATU/μg. 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 these vectors may permit sustained local antagonism of thrombin activity in vivo.

KW - Endothelial cells

KW - Gene therapy

KW - Hirudin

KW - Signal peptide

KW - Thrombin

UR - http://www.scopus.com/inward/record.url?scp=0033037277&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033037277&partnerID=8YFLogxK

U2 - 10.1038/sj.gt.3300824

DO - 10.1038/sj.gt.3300824

M3 - Article

VL - 6

SP - 385

EP - 392

JO - Gene Therapy

JF - Gene Therapy

SN - 0969-7128

IS - 3

ER -