Summary
Retroviral vectors have been used as gene delivery vehicles for more than two decades and continue to be the best available tool for stable and efficient transfer of therapeutic genes into various cell types. Although most gene therapy preclinical studies presently use crude or concentrated retroviral vector supernatants, purification to eliminate serum and host-derived impurities contained in these stocks will be a necessary requirement for clinical applications. Chromatography is deemed the most promising technology for large-scale purification of viral vectors. Heparin affinity chromatography offers the possibility to selectively and efficiently purify retroviruses. This chapter gives a simple, reproducible, and scaleable protocol for the purification of bioactive VSV-G pseudotyped retroviral vectors that employs membrane and chromatography technologies. The protocol can be easily adapted for the purification of different retroviral vector pseudotypes and lentiviral vectors. The purification techniques described here represent a significant improvement over the conventional sucrose density gradient methodology used for retrovirus purification and will hopefully contribute to the technological progress in the field of gene therapy.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Aiuti, A., Slavin, S., Aker, M., Ficara, F., Deola, S., Mortellaro, A., Morecki, S., Andolfi, G., Tabucchi, A., Carlucci, F., Marinello, E., Cattaneo, F., Vai, S., Servida, P., miniero, R., Roncarolo, M. G., and Bordignon, C. (2002) Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 296, 2410–3.
Cavazzana-Calvo, M., Hacein-Bey, S., de Saint Basile, G., Gross, F., Yvon, E., Nusbaum, P., Selz, F., Hue, C., Certain, S., Casanova, J. L., Bousso, P., Deist, F. L., and Fischer, A. (2000) Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 288, 669–72.
Gaspar, H. B., Parsley, K. L., Howe, S., King, D., Gilmour, K. C., Sinclair, J., Brouns, G., Schmidt, M., Von Kalle, C., Barington, T., Jakobsen, M. A., Christensen, H. O., Al Ghonaium, A., White, H. N., Smith, J. L., Levinsky, R. J., Ali, R. R., Kinnon, C., and Thrasher, A. J. (2004) Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet 364, 2181–7.
Gomez-Navarro, J., Curiel, D. T., and Douglas, J. T. (1999) Gene therapy for cancer. Eur J Cancer 35, 2039–57.
Rainov, N. G., and Ren, H. (2003) Clinical trials with retrovirus mediated gene therapy–what have we learned? J Neurooncol 65, 227–36.
Yamada, K., McCarty, D. M., Madden, V. J., and Walsh, C. E. (2003) Lentivirus vector purification using anion exchange HPLC leads to improved gene transfer. Biotechniques 34, 1074–8, 80.
Baekelandt, V., Eggermont, K., Michiels, M., Nuttin, B., and Debyser, Z. (2003) Optimized lentiviral vector production and purification procedure prevents immune response after transduction of mouse brain. Gene Ther 10, 1933–40.
Scherr, M., Battmer, K., Eder, M., Schule, S., Hohenberg, H., Ganser, A., Grez, M., and Blomer, U. (2002) Efficient gene transfer into the CNS by lentiviral vectors purified by anion exchange chromatography. Gene Ther 9, 1708–14.
Segura, M. M., Kamen, A., and Garnier, A. (2006) Downstream processing of oncoretroviral and lentiviral gene therapy vectors. Biotechnol Adv 24, 321–37.
Kuiper, M., Sanches, R. M., Walford, J. A., and Slater, N. K. (2002) Purification of a functional gene therapy vector derived from Moloney murine leukaemia virus using membrane filtration and ceramic hydroxyapatite chromatography. Biotechnol Bioeng 80, 445–53.
Segura, M., Kamen, A., Trudel, P., and Garnier, A. (2005) A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography. Biotechnol Bioeng 90, 391–404.
Transfiguracion, J., Jaalouk, D. E., Ghani, K., Galipeau, J., and Kamen, A. (2003) Size-exclusion chromatography purification of high-titer vesicular stomatitis virus G glycoprotein-pseudotyped retrovectors for cell and gene therapy applications. Hum Gene Ther 14, 1139–53.
Williams, S. L., Eccleston, M. E., and Slater, N. K. (2005) Affinity capture of a biotinylated retrovirus on macroporous monolithic adsorbents: towards a rapid single-step purification process. Biotechnol Bioeng 89, 783–7.
Williams, S. L., Nesbeth, D., Darling, D. C., Farzaneh, F., and Slater, N. K. (2005) Affinity recovery of Moloney Murine Leukaemia Virus. J Chromatogr B Analyt Technol Biomed Life Sci 820, 111–9.
Ye, K., Jin, S., Ataai, M. M., Schultz, J. S., and Ibeh, J. (2004) Tagging retrovirus vectors with a metal binding peptide and one-step purification by immobilized metal affinity chromatography. J Virol 78, 9820–7.
Reeves, L., and Cornetta, K. (2000) Clinical retroviral vector production: step filtration using clinically approved filters improves titers. Gene Ther 7, 1993–8.
Cruz, P. E., Goncalves, D., Almeida, J., Moreira, J. L., and Carrondo, M. J. (2000) Modeling retrovirus production for gene therapy. 2. Integrated optimization of bioreaction and downstream processing. Biotechnol Prog 16, 350–7.
Andreadis, S. T., Roth, C. M., Le Doux, J. M., Morgan, J. R., and Yarmush, M. L. (1999) Large-scale processing of recombinant retroviruses for gene therapy. Biotechnol Prog 15, 1–11.
Katane, M., Takao, E., Kubo, Y., Fujita, R., and Amanuma, H. (2002) Factors affecting the direct targeting of murine leukemia virus vectors containing peptide ligands in the envelope protein. EMBO Rep 3, 899–904.
Palù, G., Parolin, C., Takeuchi, Y., and Pizzato, M. (2000) Progress with retroviral gene vectors. Rev Med Virol 10, 185–202.
Tai, C. K., Logg, C. R., Park, J. M., Anderson, W. F., Press, M. F., and Kasahara, N. (2003) Antibody-mediated targeting of replication-competent retroviral vectors. Hum Gene Ther 14, 789–802.
Navarro del Cañizo, A. A., Mazza, M., Bellinzoni, R., and Cascone, O. (1996) Foot and mouth disease virus concentration and purification by affinity chromatography. Appl Biochem Biotechnol 61, 399–409.
O’Keeffe, R. S., Johnston, M. D., and Slater, N. K. (1999) The affinity adsorptive recovery of an infectious herpes simplex virus vaccine. Biotechnol Bioeng 62, 537–45.
Zolotukhin, S., Byrne, B. J., Mason, E., Zolotukhin, I., Potter, M., Chesnut, K., Summerford, C., Samulski, R. J., and Muzyczka, N. (1999) Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Ther 6, 973–85.
Burnouf, T., and Radosevich, M. (2001) Affinity chromatography in the industrial purification of plasma proteins for therapeutic use. J Biochem Biophys Methods 49, 575–86.
McTaggart, S., and Al-Rubeai, M. (2000) Effects of culture parameters on the production of retroviral vectors by a human packaging cell line. Biotechnol Prog 16, 859–65.
Wikstrom, K., Blomberg, P., and Islam, K. B. (2004) Clinical grade vector production: analysis of yield, stability, and storage of gmp-produced retroviral vectors for gene therapy. Biotechnol Prog 20, 1198–203.
Acknowledgments
The authors thank Normand Arcand and Alice Bernier for helpful discussions and Gavin Whissell for careful review of this manuscript. This work was supported by a NSERC Strategic Project grant and the NCE Canadian Stem Cell Network.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
de las Mercedes Segura, M., Kamen, A., Garnier, A. (2008). Purification of Retrovirus Particles Using Heparin Affinity Chromatography. In: Le Doux, J.M. (eds) Gene Therapy Protocols. Methods in Molecular Biology™, vol 434. Humana Press. https://doi.org/10.1007/978-1-60327-248-3_1
Download citation
DOI: https://doi.org/10.1007/978-1-60327-248-3_1
Publisher Name: Humana Press
Print ISBN: 978-1-60327-247-6
Online ISBN: 978-1-60327-248-3
eBook Packages: Springer Protocols