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Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications

Abstract

In recent years, recombinant adenoviral and adeno-associated viral (AAV) vectors have been exploited in a number of gene delivery approaches. The use of these vectors in clinical gene transfer has increased the demand for their characterization, production and purification. Although the classical method of adenovirus or AAV purification by density gradient centrifugation is effective on a small scale, chromatographic separation is the most versatile and powerful method for large-scale production of recombinant adenovirus or AAV. This review describes different chromatographic modes for adenovirus or AAV purification and process development, as well as the utility of different purification steps for virus production. Advances in the development of viral vectors for gene therapy, such as the discovery of new AAV serotypes, adenoviral and AAV retargeting and improved production of helper-dependent adenoviral vectors, require further development of efficient purification methods.

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References

  1. http://www.wiley.co.uk/genetherapy/clinical/. A Journal of Gene Medicine website.

  2. Green M et al. Adenovirus DNA I. Molecular weight and conformation. PNAS USA 1967; 57: 1302–1309.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lonberg-Holm K, Crowell RL, Philipson L . Unrelated animal viruses share receptors. Nature 1976; 259: 679–681.

    Article  CAS  PubMed  Google Scholar 

  4. St George JA . Gene therapy progress and prospects: adenoviral vectors. Gene Therapy 2003; 10: 1135–1141.

    Article  CAS  PubMed  Google Scholar 

  5. Volpers C, Kochanek S . Adenoviral vectors for gene transfer and therapy. J Gene Med 2004; 6 (Suppl 1): S164–S171.

    Article  CAS  PubMed  Google Scholar 

  6. Danthinne X, Imperiale MJ . Production of first generation adenovirus vectors: a review. Gene Therapy 2000; 7: 1707–1714.

    Article  CAS  PubMed  Google Scholar 

  7. Berns KI . Parvovirus replication. Microbiol Rev 1990; 54: 316–329.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Siegl G et al. Characteristics and taxonomy of Parvoviridae. Intervirology 1985; 23: 61–73.

    Article  CAS  PubMed  Google Scholar 

  9. Flotte TR . Gene therapy progress and prospects: recombinant adeno-associated virus (rAAV) vectors. Gene Therapy 2004; 11: 805–810.

    Article  CAS  PubMed  Google Scholar 

  10. Monahan PE, Samulski RJ . AAV vectors: is clinical success on the horizon? Gene Therapy 2000; 7: 24–30.

    Article  CAS  PubMed  Google Scholar 

  11. Jooss K, Yang Y, Fisher KJ, Wilson JM . Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers. J Virol 1998; 72: 4212–4223.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Grimm D, Kleinschmidt JA . Progress in adeno-associated virus type 2 vector production: promises and prospects for clinical use. Hum Gene Ther 1999; 10: 2445–2450.

    Article  CAS  PubMed  Google Scholar 

  13. Hermonat PL, Muzyczka N . Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc Natl Acad Sci USA 1984; 81: 6466–6470.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Tratschin JD, Miller IL, Smith MG, Carter BJ . Adeno-associated virus vector for high-frequency integration, expression, and rescue of genes in mammalian cells. Mol Cell Biol 1985; 5: 3251–3260.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. McLaughlin SK, Collis P, Hermonat PL, Muzyczka N . Adeno-associated virus general transduction vectors: analysis of proviral structures. J Virol 1988; 62: 1963–1973.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Xiao X, Li J, Samulski RJ . Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus. J Virol 1998; 72: 2224–2232.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Blanche F et al. An improved anion-exchange HPLC method for the detection and purification of adenoviral particles. Gene Therapy 2000; 7: 1055–1062.

    Article  CAS  PubMed  Google Scholar 

  18. Huyghe BG et al. Purification of a type 5 recombinant adenovirus encoding human p53 by column chromatography. Hum Gene Ther 1995; 6: 1403–1416.

    Article  CAS  PubMed  Google Scholar 

  19. Green AP et al. A new scalable method for the purification of recombinant adenovirus vectors. Hum Gene Ther 2002; 13: 1921–1934.

    Article  CAS  PubMed  Google Scholar 

  20. Clark KR, Liu X, McGrath JP, Johnson PR . Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses. Hum Gene Ther 1999; 10: 1031–1039.

    Article  CAS  PubMed  Google Scholar 

  21. Auricchio A et al. Isolation of highly infectious and pure adeno-associated virus type 2 vectors with a single-step gravity-flow column. Hum Gene Ther 2001; 12: 71–76.

    Article  CAS  PubMed  Google Scholar 

  22. Graham FL, Smiley J, Russell WC, Nairn R . Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol 1977; 36: 59–74.

    Article  CAS  PubMed  Google Scholar 

  23. Fallaux FJ et al. New helper cells and matched early region 1-deleted adenovirus vectors prevent generation of replication-competent adenoviruses. Hum Gene Ther 1998; 9: 1909–1917.

    Article  CAS  PubMed  Google Scholar 

  24. Schiedner G, Hertel S, Kochanek S . Efficient transformation of primary human amniocytes by E1 functions of Ad5: generation of new cell lines for adenoviral vector production. Hum Gene Ther 2000; 11: 2105–2116.

    Article  CAS  PubMed  Google Scholar 

  25. Gao GP, Engdahl RK, Wilson JM . A cell line for high-yield production of E1-deleted adenovirus vectors without the emergence of replication-competent virus. Hum Gene Ther 2000; 11: 213–219.

    Article  CAS  PubMed  Google Scholar 

  26. Kochanek S, Schiedner G, Volpers C . High-capacity ‘gutless’ adenoviral vectors. Curr Opin Mol Ther 2001; 3: 454–463.

    CAS  PubMed  Google Scholar 

  27. Kochanek S et al. A new adenoviral vector: replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase. Proc Natl Acad Sci USA 1996; 93: 5731–5736.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Parks RJ et al. A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci USA 1996; 93: 13565–13570.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Umana P et al. Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination. Nat Biotechnol 2001; 19: 582–585.

    Article  CAS  PubMed  Google Scholar 

  30. Samulski RJ, Chang LS, Shenk T . A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication. J Virol 1987; 61: 3096–3101.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Samulski RJ, Chang LS, Shenk T . Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression. J Virol 1989; 63: 3822–3828.

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Grimm D, Kern A, Rittner K, Kleinschmidt JA . Novel tools for production and purification of recombinant adenoassociated virus vectors. Hum Gene Ther 1998; 9: 2745–2760.

    Article  CAS  PubMed  Google Scholar 

  33. Salvetti A et al. Factors influencing recombinant adeno-associated virus production. Hum Gene Ther 1998; 9: 695–706.

    Article  CAS  PubMed  Google Scholar 

  34. Chadeuf G et al. Efficient recombinant adeno-associated virus production by a stable rep–cap HeLa cell line correlates with adenovirus-induced amplification of the integrated rep–cap genome. J Gene Med 2000; 2: 260–268.

    Article  CAS  PubMed  Google Scholar 

  35. Mathews LC, Gray JT, Gallagher MR, Snyder RO . Recombinant adeno-associated viral vector production using stable packaging and producer cell lines. Methods Enzymol 2002; 346: 393–413.

    Article  CAS  PubMed  Google Scholar 

  36. Gao G et al. Purification of recombinant adeno-associated virus vectors by column chromatography and its performance in vivo. Hum Gene Ther 2000; 11: 2079–2091.

    Article  CAS  PubMed  Google Scholar 

  37. Saudan P, Vlach J, Beard P . Inhibition of S-phase progression by adeno-associated virus Rep78 protein is mediated by hypophosphorylated pRb. EMBO J 2000; 19: 4351–4361.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Schmidt M, Afione S, Kotin RM . Adeno-associated virus type 2 Rep78 induces apoptosis through caspase activation independently of p53. J Virol 2000; 74: 9441–9450.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Clark KR, Voulgaropoulou F, Fraley DM, Johnson PR . Cell lines for the production of recombinant adeno-associated virus. Hum Gene Ther 1995; 6: 1329–1341.

    Article  CAS  PubMed  Google Scholar 

  40. Gao GP et al. High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus. Hum Gene Ther 1998; 9: 2353–2362.

    Article  CAS  PubMed  Google Scholar 

  41. Qiao C et al. A novel gene expression control system and its use in stable, high-titer 293 cell-based adeno-associated virus packaging cell lines. J Virol 2002; 76: 13015–13027.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Johnston KM et al. HSV/AAV hybrid amplicon vectors extend transgene expression in human glioma cells. Hum Gene Ther 1997; 8: 359–370.

    Article  CAS  PubMed  Google Scholar 

  43. Urabe M, Ding C, Kotin RM . Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum Gene Ther 2002; 13: 1935–1943.

    Article  CAS  PubMed  Google Scholar 

  44. Smith RH, Ding C, Kotin RM . Serum-free production and column purification of adeno-associated virus type 5. J Virol Methods 2003; 114: 115–124.

    Article  CAS  PubMed  Google Scholar 

  45. Kamen A, Henry O . Development and optimization of an adenovirus production process. J Gene Med 2004; 6 (Suppl 1): S184–S192.

    Article  CAS  PubMed  Google Scholar 

  46. Drittanti L et al. Optimised helper virus-free production of high-quality adeno-associated virus vectors. J Gene Med 2001; 3: 59–71.

    Article  CAS  PubMed  Google Scholar 

  47. O'Riordan CR, Lachapelle AL, Vincent KA, Wadsworth SC . Scaleable chromatographic purification process for recombinant adeno-associated virus (rAAV). J Gene Med 2000; 2: 444–454.

    Article  CAS  PubMed  Google Scholar 

  48. Anderson R et al. A method for the preparation of highly purified adeno-associated virus using affinity column chromatography, protease digestion and solvent extraction. J Virol Methods 2000; 85: 23–34.

    Article  CAS  PubMed  Google Scholar 

  49. Potter M et al. Streamlined large-scale production of recombinant adeno-associated virus (rAAV) vectors. Methods Enzymol 2002; 346: 413–430.

    Article  CAS  PubMed  Google Scholar 

  50. Zolotukhin S et al. Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield. Gene Therapy 1999; 6: 973–985.

    Article  CAS  PubMed  Google Scholar 

  51. Green M, Pina M . Biochemical studies on adenovirus multiplication. VI. Properties of highly purified tumorigenic human adenoviruses and their DNA's. Proc Natl Acad Sci USA 1964; 51: 1251–1259.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Zolotukhin S et al. Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors. Methods 2002; 28: 158–167.

    Article  CAS  PubMed  Google Scholar 

  53. Stewart PL, Burnett RM . Adenovirus structure by X-ray crystallography and electron microscopy. Curr Top Microbiol Immunol 1995; 199 (Part 1): 25–38.

    PubMed  Google Scholar 

  54. Brument N et al. A versatile and scalable two-step ion-exchange chromatography process for the purification of recombinant adeno-associated virus serotypes-2 and -5. Mol Ther 2002; 6: 678–686.

    Article  CAS  PubMed  Google Scholar 

  55. Kaludov N, Handelman B, Chiorini JA . Scalable purification of adeno-associated virus type 2, 4, or 5 using ion-exchange chromatography. Hum Gene Ther 2002; 13: 1235–1243.

    Article  CAS  PubMed  Google Scholar 

  56. Tamayose K, Hirai Y, Shimada T . A new strategy for large-scale preparation of high-titer recombinant adeno-associated virus vectors by using packaging cell lines and sulfonated cellulose column chromatography. Hum Gene Ther 1996; 7: 507–513.

    Article  CAS  PubMed  Google Scholar 

  57. Summerford C, Samulski RJ . Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions. J Virol 1998; 72: 1438–1445.

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Schnepp B, Clark KR . Highly purified recombinant adeno-associated virus vectors. Preparation and quantitation. Methods Mol Med 2002; 69: 427–443.

    CAS  PubMed  Google Scholar 

  59. Rabinowitz JE et al. Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. J Virol 2002; 76: 791–801.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Kaludov N et al. Adeno-associated virus serotype 4 (AAV4) and AAV5 both require sialic acid binding for hemagglutination and efficient transduction but differ in sialic acid linkage specificity. J Virol 2001; 75: 6884–6893.

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Walters RW et al. Binding of adeno-associated virus type 5 to 2,3-linked sialic acid is required for gene transfer. J Biol Chem 2001; 276: 20610–20616.

    Article  CAS  PubMed  Google Scholar 

  62. Auricchio A, O'Connor E, Hildinger M, Wilson JM . A single-step affinity column for purification of serotype-5 based adeno-associated viral vectors. Mol Ther 2001; 4: 372–374.

    Article  CAS  PubMed  Google Scholar 

  63. Di Pasquale G et al. Identification of PDGFR as a receptor for AAV-5 transduction. Nat Med 2003; 9: 1306–1312.

    Article  CAS  PubMed  Google Scholar 

  64. Andreadis ST et al. Large-scale processing of recombinant retroviruses for gene therapy. Biotechnol Prog 1999; 15: 1–11.

    Article  CAS  PubMed  Google Scholar 

  65. Crooks AJ, Lee JM, Dowsett AB, Stephenson JR . Purification and analysis of infectious virions and native non-structural antigens from cells infected with tick-borne encephalitis virus. J Chromatogr 1990; 502: 59–68.

    Article  CAS  PubMed  Google Scholar 

  66. Shabram PW et al. Analytical anion-exchange HPLC of recombinant type-5 adenoviral particles. Hum Gene Ther 1997; 8: 453–465.

    Article  CAS  PubMed  Google Scholar 

  67. Klyushnichenko V, Bernier A, Kamen A, Harmsen E . Improved high-performance liquid chromatographic method in the analysis of adenovirus particles. J Chromatogr B 2001; 755: 27–36.

    Article  CAS  Google Scholar 

  68. Transfiguracion J et al. Validation of a high-performance liquid chromatographic assay for the quantification of adenovirus type 5 particles. J Chromatogr B 2001; 761: 187–194.

    Article  CAS  Google Scholar 

  69. Vellekamp G et al. Empty capsids in column-purified recombinant adenovirus preparations. Hum Gene Ther 2001; 12: 1923–1936.

    Article  CAS  PubMed  Google Scholar 

  70. Debelak D et al. Cation-exchange high-performance liquid chromatography of recombinant adeno-associated virus type 2. J Chromatogr B 2000; 740: 195–202.

    Article  CAS  Google Scholar 

  71. Mittereder N, March KL, Trapnell BC . Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J Virol 1996; 70: 7498–7509.

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Lehmberg E et al. Reversed-phase high-performance liquid chromatographic assay for the adenovirus type 5 proteome. J Chromatogr B 1999; 732: 411–423.

    Article  CAS  Google Scholar 

  73. Schnell MA et al. Activation of innate immunity in nonhuman primates following intraportal administration of adenoviral vectors. Mol Ther 2001; 3: 708–722.

    Article  CAS  PubMed  Google Scholar 

  74. Wickham TJ, Roelvink PW, Brough DE, Kovesdi I . Adenovirus targeted to heparan-containing receptors increases its gene delivery efficiency to multiple cell types. Nat Biotechnol 1996; 14: 1570–1573.

    Article  CAS  PubMed  Google Scholar 

  75. Douglas JT et al. A system for the propagation of adenoviral vectors with genetically modified receptor specificities. Nat Biotechnol 1999; 17: 470–475.

    Article  CAS  PubMed  Google Scholar 

  76. Parrott MB et al. Metabolically biotinylated adenovirus for cell targeting, ligand screening, and vector purification. Mol Ther 2003; 8: 688–700.

    Article  CAS  PubMed  Google Scholar 

  77. Sandig V et al. Optimization of the helper-dependent adenovirus system for production and potency in vivo. Proc Natl Acad Sci USA 2000; 97: 1002–1007.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Palmer D, Ng P . Improved system for helper-dependent adenoviral vector production. Mol Ther 2003; 8: 846–852.

    Article  CAS  PubMed  Google Scholar 

  79. Miao CH et al. Nonrandom transduction of recombinant adeno-associated virus vectors in mouse hepatocytes in vivo: cell cycling does not influence hepatocyte transduction. J Virol 2000; 74: 3793–3803.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Grimm D . Research advances in virology. In: Samal SK, Liebler-Tenorio E, Delsert C, Paton D (eds). Adeno-Associated Virus (AAV) Serotypes as Vectors for Human Gene Therapy. Puthen Chalai, Global Research Network: India, 2000, pp 91–114.

    Google Scholar 

  81. Gao GP et al. Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc Natl Acad Sci USA 2002; 99: 11854–11859.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Chao H et al. Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors. Mol Ther 2000; 2: 619–623.

    Article  CAS  PubMed  Google Scholar 

  83. Hildinger M et al. Hybrid vectors based on adeno-associated virus serotypes 2 and 5 for muscle-directed gene transfer. J Virol 2001; 75: 6199–6203.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Buning H et al. Receptor targeting of adeno-associated virus vectors. Gene Therapy 2003; 10: 1142–1151.

    Article  CAS  PubMed  Google Scholar 

  85. Wu P et al. Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism. J Virol 2000; 74: 8635–8647.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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We thank John Rudge, Simon Hoffenberg and Christopher Daly for their helpful comments and suggestions.

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Burova, E., Ioffe, E. Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications. Gene Ther 12 (Suppl 1), S5–S17 (2005). https://doi.org/10.1038/sj.gt.3302611

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