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Saporin Suicide Gene Therapy

  • Natasa Zarovni
  • Riccardo Vago
  • Maria Serena FabbriniEmail author
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 542)

Summary

New genes useful in suicide gene therapy are those encoding toxins such as plant ribosome-inactivating proteins (RIPs), which can irreversibly block protein synthesis, triggering apoptotic cell death. Plasmids expressing a cytosolic saporin (SAP) gene from common soapwort (Saponaria officinalis) are generated by placing the region encoding the mature plant toxin under the control of strong viral promoters and may be placed under tumor-specific promoters. The ability of the resulting constructs to inhibit protein synthesis is tested in cultured tumor cells co-transfected with a luciferase reporter gene. SAP expression driven by the cytomegalovirus (CMV) promoter (pCI-SAP) demonstrates that only 10 ng of plasmid DNA per 1.6 × 104 B16 melanoma cells drastically reduces luciferase reporter activity to 18% of that in control cells (1). Direct intratumoral injections are performed in an aggressive melanoma model. B16 melanoma-bearing mice injected with pCI-SAP complexed with lipofectamine or N-(2,3-dioleoyloxy-1-propyl) trimethylammonium methyl sulfate (DOTAP) show a noteworthy attenuation in tumor growth, and this effect is significantly augmented by repeated administrations of the DNA complexes. Here, we describe in detail this cost-effective and safe suicide gene approach.

Key Words

DOTAP melanoma nonviral vectors polyethylenimine (PEI) ribosome-inactivating proteins ricin saporin suicide gene therapy 

Notes

Acknowledgments

The authors are deeply indebted to Lucia Monaco for the development of the nonviral vector strategy, the pCISfiLuc reporter gene construct, and especially for her advice and continuous support. This work has been partially supported by the Italian Cancer Research Association (AIRC, grant 131/01) and by IBBA-CNR, Milano.

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Natasa Zarovni
    • 1
  • Riccardo Vago
    • 1
  • Maria Serena Fabbrini
    • 2
    Email author
  1. 1.Department of Biological and Technological Research, DibitSan Raffaele H Scientific InstituteMilanoItaly
  2. 2.Istituto di Biologia e Biotecnologia AgrariaConsiglio Nazionale delle Ricerche, IBBA-CNR via BassiniMilano

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