Abstract
For many gene therapy applications the effective titre of retroviral vectors is a limiting factor both in vitro and in vivo. Purification and concentration of retrovirus from packaging cell supernatant can overcome this problem. To this end we have investigated a novel procedure which involves complexing retrovirus to a dense and particulate substrate followed by a short low-speed centrifugation. The study reported here uses heat-killed, formaldehyde fixed Staphylococcus aureus (Pansorbin) absorbed to PG13 derived retrovirus. This complex was then used to harvest retrovirus from packaging cell supernatant: centrifugation and washing of this complex allows the retrovirus to be both purified and concentrated. This procedure increases the effective titre of retrovirus by up to 7500-fold after an only 200-fold reduction in volume. The affinity of Pansorbin for retrovirus allows concentration regardless of its encoded genes and makes this protocol applicable to other popular packaging cells and envelope proteins. Possible explanations for the marked increase in titre of concentrated virus and the mechanism governing the complexing of retrovirus to Pansorbin are discussed.
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References
Miller AD, Rosman GJ . Improved retroviral vectors for gene transfer and expression Biotechniques 1989 8: 980–990
Riviere I, Brose K, Mulligan RC . Effects of retroviral design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells Proc Natl Acad Sci USA 1995 92: 6733–6737
Miller AD et al. Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus J Virol 1991 65: 2220–2224
Ory DS, Neugeboren BA, Mulligan RC . A stable human-derived packaging cell line for the production of high titre retrovirus/vesicular stomatitis virus G pseudotypes Proc Natl Acad Sci USA 1996 93: 11400–11406
Cosset F-L et al. High-titer packaging cells producing recombinant retroviruses resistant to human serum J Virol 1995 69: 7430–7436
Burns JC et al. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to a very high titre and efficient gene transfer into mammalian and nonmammalian cells Proc Natl Acad Sci USA 1993 90: 8033–8037
Kozak SL, Kabat D . Ping-pong amplification of a retroviral vector achieves high-level gene expression: human growth hormone production J Virol 1990 64: 3500–3508
Parente MK, Wolfe JH . Production of increased titer retrovirus vectors from stable producer cell lines by superinfection and concentration Gene Therapy 1996 3: 756–760
Kotani H et al. Improved methods of retroviral vector transduction and production for gene therapy Hum Gene Ther 1994 5: 19–28
Bunnell BA et al. High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes Proc Natl Acad Sci USA 1995 92: 7739–7743
Olsen JC, Sechelski J . Use of sodium butyrate to enhance production of retroviral vectors expressing CTFR cDNA Hum Gene Ther 1995 6: 1195–1202
Manning JS, Hackett AJ, Darby NB Jr . Effect of polycations on sensitivity of balb/3T3 cells to murine leukaemia and sarcoma virus infectivity Appl Microbiol 1971 22: 1162–1163
Toyoshima K, Vogt PK . Enhancement and inhibition of avian sarcoma viruses by polycations and polyanions Virology 1969 38: 414–426
Porter CD et al. Cationic liposomes enhance the rate of transduction by a recombinant retroviral vector in vitro and in vivo J Virol 1998 72: 4832–4840
Chuck AS, Clarke MF, Palson BO . Retroviral infection is limited by brownian motion Hum Gene Ther 1996 7: 1527–1534
Hanenberg H et al. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells Nature Med 1996 2: 876–882
Paul RW et al. Increased viral titer through concentration of viral harvests from retroviral packaging lines Hum Gene Ther 1993 4: 609–615
Miller DL, Meikle PJ, Anson DS . A rapid and efficient method for concentration of small volumes of retroviral supernatant Nucleic Acids Res 1996 24: 1576–1577
Chu T-HT, Dornburg R . Toward highly efficient cell-type-specific gene transfer with retroviral vectors displaying single-chain antibodies J Virol 1997 71: 720–725
Olsen JC, Johnson LG, Yankaskas JR . Methods for the use of retroviral vectors for transfer of the CTFR gene to airway epithelium. In: Robbins P (ed) Gene Therapy Protocols Humana Press: New Jersey 1997 pp 153–168
Morling FJ, Russell SJ . Enhanced transduction efficiency of retroviral vectors coprecipitated with calcium phosphate Gene Therapy 1995 2: 504–508
LeDoux JM, Morgan JR, Snow RG, Yarmush ML . Proteoglycans secreted by packaging cell lines inhibit retrovirus infection J Virol 1996 70: 6468–6473
Russell SJ . Making high titre retroviral producer cells. In: Collins MKL (ed) Methods in Molecular Biology: Vol 8 Practical Molecular Virology Humana Press: New Jersey 1991 pp 29–48
Bowles NE et al. A simple and efficient method for the concentration and purification of recombinant retrovirus for increased hepotocyte transduction in vivo Hum Gene Ther 1996 7: 1735–1742
Seppen J, Kimmel RJ, Osborne WRA . Serum-free production, concentration and purification of recombinant retroviruses Biotechniques 1997 23: 788–790
McNeish IA et al. Virus directed enzyme prodrug therapy for ovarian and pancreatic cancer using retrovirally delivered E. coli nitroreductase and CB1954 Gene Therapy 1998 5: 1061–1069
Liu M-L, Winther BL, Kay MA . Pseudotransduction of hepatocytes by using concentrated pseudotyped vesicular stomatitis virus G glycoprotein (VSV-G)-moloney murine leukemia virus-derived retrovirus vectors: comparison of VSV-G and amphotropic vectors for hepatic gene transfer J Virol 1996 70: 2497–2502
Palson B, Andreadis S . The physio-chemical factors that govern retrovirus-mediated gene transfer Exp Hematol 1997 25: 94–102
Morgan JR et al. Retrovirus infection – effect of time and target-cell number J Virol 1995 69: 6994–7000
Gäken J et al. Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron (submitted)
Dardalhon V et al. Green fluorescent protein as a selectable marker of fibronectin-facilitated retroviral gene transfer in primary human T-lymphocytes Hum Gene Ther 1999 10: 5–14
Signas C et al. Nucleotide sequence of the gene for fibronectin-binding protein from Staphylococcus aureus: use of this peptide sequence in the synthesis of biologically active peptides Proc Natl Acad Sci USA 1989 86: 699–703
Jonsson K et al. Two different genes encode fibronectin binding proteins in Staphylococcus aureus: the complete nucleotide sequence and characterization of the second gene Eur J Biochem 1991 202: 1041–1048
Shantz EM . PANSORBIN, Staphylococcus aureus cells: a review and bibliography of the immunological applications of fixed protein A-bearing Staphylococcus aureus cells. Calbiochem 1994
Freeman GT et al. B7, a new member of the IG superfamily with unique expression on activated and neoplastic B-cells J Immunol 1989 143: 2714–2722
Spear GT et al. Host cell-derived complement proteins CD55 and CD59 are incorporated into the virions of 2 unrelated enveloped viruses – human T-cell leukemia/lymphoma virus type-1 (HTLV-1) and human cytomegalovirus (HCMV) J Immunol 1995 155: 4376–4381
Balliet JW, Bates P . Efficient infection mediated by viral receptors incorporated into retroviral particles J Virol 1998 72: 671–676
Izumi M et al. Blasticidin S-resistance gene (bsr): a novel selectable marker for mammalian cells Exp Cell Res 1991 197: 229–233
Markowitz D, Goff S, Bank A . Construction and use of a safe and efficient amphotropic packaging cell line Virology 1988 167: 400–406
Miller AD, Buttimore C . Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production Mol Cell Biol 1986 6: 2895–2902
Markowitz D, Goff S, Bank A . A safe packaging line for gene transfer: separating viral genes on two different plasmids J Virol 1988 62: 1120–1124
Kayman SC et al. The hypervariable domain of the murine leukaemia virus surface protein tolerates large insertions and deletions, enabling developement of a retroviral particle display system J Virol 1999 73: 1802–1808
Daley GQ, Baltimore D . Transformation of an interleukin 3-dependent haematopoietic cell line by the chronic myelogenous leukemia-specific P210bcr/abl protein Proc Natl Acad Sci USA 1988 85: 9312–9316
Vercellotti GM et al. Extracellular matrix proteins (Fibronectin, Laminin, and Type IV Collagen) bind and aggregate bacteria Am J Pathol 1985 120: 13–21
Jordan-Sciutto KL et al. Reduction in fibronectin expression and alteration in cell morphology are coincident in NIH3T3 cells expressing a mutant E2F1 transcription factor Exp Cell Res 1997 236: 527–536
Dean DC, Newby RF, Bourgeois S . Regulation of fibronectin biosynthesis by dexamethasone, transforming growth factor-beta, and cAMP in human cell-lines J Cell Biol 1988 106: 2159–2170
Gerin PA et al. Improved titers of retroviral vectors from the human FLYRD18 packaging cell line in serum- and protein-free medium Hum Gene Ther 1999 10: 1965–1974
Touhami M, Bourge J-F, Legrand C . Increased adhesion of the promyelocyte leukaemia cell line, NB4, to fibronectin and thrombospondin upon all-trans-retinoic acid treatment Br J Haematol 1999 104: 706–714
Bansal SC et al. Ex vivo removal of serum IgG in a patient with colon carcinoma. Some biochemical, immunological and histological observations Cancer 1978 42: 1–18
Cowan FM . Staphylococcus Aureus protein A immunoadsorption: the rationale for systemic protein A immunotherapy Toxicol Meth 1997 7: 9–15
Steele G, Ankerst J, Sjogren HO . Alteration of in vitro anti-tumour activity of tumour-bearer sera by adsorption with Staphylococcus aureus Int J Cancer 1974 14: 83–92
Fennelly DW et al. A phase II trial of extracorporeal plasma immunoadsorption of patient plasma with PROSORBA columns for treating metastatic breast cancer Cancer 1995 75: 2099–2102
Messerschmidt GL et al. Protein A immunotherapy in the treatment of cancer: an update Semin Hematol 1989 26 (Suppl. 1): 19–24
Engelman RW, Good RA, Day NK . Clearance of retroviremia and regression of malignancy in cats with leukemia–lymphoma during treatment with staphylococcal protein A Cancer Detect Prev 1987 10: 435–444
Snyder HW, Reed DE, Jones FR . Remission of FeLV-associated lymphosarcoma and persistent viral infection after extracorporeal immunoadsorption of plasma using staphylococcal protein A columns: details of immune response Semin Hematol 1989 26 (Suppl. 1): 25–30
Acknowledgements
This work was supported by grant 9605 from the Leukaemia Research Fund and The Lewis Family Research Trust. We thank Professor MKL Collins, Department of Immunology, Windeyer Institute of Medical Sciences, University College London for the gift of the FLYA13 and FLYRD18 retroviral packaging cell lines.
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Darling, D., Hughes, C., Galea-Lauri, J. et al. Low-speed centrifugation of retroviral vectors absorbed to a particulate substrate: a highly effective means of enhancing retroviral titre. Gene Ther 7, 914–923 (2000). https://doi.org/10.1038/sj.gt.3301201
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DOI: https://doi.org/10.1038/sj.gt.3301201
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