Browse > Article
http://dx.doi.org/10.5352/JLS.2012.22.6.703

Improved Production Efficiencies of Various Adeno-Associated Virus (AAV) Serotypes and a Novel Universal AAV Titration Method  

Cho, Young-Hwa (Juseong Gene Therapy R&D Center, Juseong University)
Choi, Ye-Jin (Biologics Research Division, National Institute of Food and Drug Safety Evaluation, KFDA)
Yun, Jung-Hee (Juseong Gene Therapy R&D Center, Juseong University)
Kim, Nam-Hee (Biologics Research Division, National Institute of Food and Drug Safety Evaluation, KFDA)
Choi, Mi-Ra (Biologics Research Division, National Institute of Food and Drug Safety Evaluation, KFDA)
Choi, Young-Kook (Juseong Gene Therapy R&D Center, Juseong University)
Kim, Kyung-Hee (Juseong Gene Therapy R&D Center, Juseong University)
Lee, Young-Ill (School of Engineering, University of Suwon)
Lee, Beom-Jun (College of Veterinary Medicine and Research Institute of Veterinary Medicine, Chungbuk National University)
Park, Kee-Rang (Juseong Gene Therapy R&D Center, Juseong University)
Publication Information
Journal of Life Science / v.22, no.6, 2012 , pp. 703-712 More about this Journal
Abstract
Adeno-associated virus (AAV) has been considered to be a very safe and efficient gene delivery system. However, the major obstacles to therapeutic usage of AAV have been to achieve highly efficient and reproducible production processes, and also to develop a reliable quantifying method of various serotypes with a simple protocol. We compared the efficiency of the conventional production protocol of AAV2 and adenovirus (Ad) co-infection to that of a new method containing AAV2 infection followed by pHelper transfection. We tested HEK293 and 293T, and further examined the time-dependent changes of AAV2 production. The new method of AAV2 and pHelper DNA gave about ten times higher production efficiency than that of the conventional protocol. The highest production efficiency in 293T was achieved as $1.61{\times}10^5$ virus genomes (v.g.)/cell by the new method of 10 MOI of AAV2 infection and 5 days post-infection. This protocol of the highest efficiency was then applied to produce various AAV serotypes and showed the efficiencies higher than $10^5$ v.g./cell. Next, we designed the universal PCR primers of highly conserved regions for various AAV serotypes to develop a simple and reliable titration method. The universal primers could amplify all the tested AAV serotypes with similar sensitivities by ten molecular copies. Therefore, this pair of universal primers can be further utilized to detect AAV contaminants in therapeutic adenoviral vectors.
Keywords
Adeno-associated virus; AAV; gene therapy; scale-up production; virus titration;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Atchison, R. W., Casto, B. C. and Hammon, W. M. 1965. Adenovirus-Associated Defective Virus Particles. Science 149, 754-756.   DOI
2 Aucoin, M. G., Perrier, M. and Kamen, A. A. 2008. Critical assessment of current adeno-associated viral vector production and quantification methods. Biotechnol. Adv. 26, 73-88.   DOI
3 Bantel-Schaal, U. and zur Hausen, H. 1984. Characterization of the DNA of a defective human parvovirus isolated from a genital site. Virology 134, 52-63.   DOI
4 Blouin, V., Brument, N., Toublanc, E., Raimbaud, I., Moullier, P. and Salvetti, A. 2004. Improving rAAV production and purification: towards the definition of a scalable process. J. Gene Med. 6, S223-228.   DOI
5 Buning, H., Perabo, L., Coutelle, O., Quadt-Humme, S. and Hallek, M. 2008. Recent developments in adeno-associated virus vector technology. J. Gene Med. 10, 717-733.   DOI
6 Chen, C. and Okayama, H. 1987. High-efficiency transformation of mammalian cells by plasmid DNA. Mol. Cell Biol. 7, 2745-2752.
7 Cho, Y. H., Park, H., Cho, E. S., Kim, W. J., Kang, B. S., Park, B. Y., Kim, Y. J., Lee, Y. I., Chang, S. I. and Park, K. 2007. A novel way of therapeutic angiogenesis using an adeno-associated virus-mediated angiogenin gene transfer. Exp. Mol. Med. 39, 412-418.   DOI   ScienceOn
8 Clark, K. R., Voulgaropoulou, F., Fraley, D. M. and Johnson, P. R. 1995. Cell lines for the production of recombinant adeno- associated virus. Hum. Gene Ther. 6, 1329-1341.   DOI
9 Clark, K. R. 2002. Recent advances in recombinant adeno- associated virus vector production. Kidney Int. 61, S9-15.   DOI
10 Collaco, R. F., Cao, X. and Trempe, J. P. 1999. A helper virus- free packing system for recombinant adeno-associated virus vectors. Gene 238, 397-405.   DOI
11 Gao, G. P., Wilson, J. M. and Wivel, N. A. 2000. Production of recombinant adeno-associated virus. Adv. Virus Res. 55, 529-543   DOI
12 Georg-Fries, B., Biederlack, S., Wolf, J. and zur Hausen, H. 1984. Analysis of proteins, helper dependence, and seroepidemiology of a new human parvovirus. Virology 134, 64-71.   DOI
13 Grimm, D., Kern, A., Rittner, K. and Kleinschmidt, J. A. 1998. Novel tools for production and purification of recombinant adeno-associated virus vectors. Hum. Gene Ther. 10, 2745-2760.
14 Grimm, D. and Kleinschmidt, J. A. 1999. Progress in adeno- associated virus type 2 vector production: promises and prospects for clinical use. Hum. Gene Ther. 10, 2445-2450.   DOI
15 Hoggan, M. D., Blacklow, N. R. and Rowe, W. P. 1966. Studies of small DNA viruses found in various adenovirus preparations: physical, biological, and immunological characteristics. Proc. Natl. Acad. Sci. USA 55, 1467-1474.   DOI
16 Kay, M. A., Manno, C. S., Ragni, M. V., Larson, P. J., Couto, L. B., McClelland, A., Glader, B., Chew, A. J., Tai, S. J., Herzog, R. W., Arruda, V., Johnson, F., Scallan, C., Skarsgard, E., Flake, A. W. and High, K. A. 2000. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat. Genet. 24, 257-261.   DOI
17 Matthews, L. C., Gray, J. T., Gallagher, M. R. and Snyder, R. O. 2002. Recombinant adeno-associated viral vector production using stable packaging and producer cell lines. Methods Enzymol. 346, 393-413.   DOI
18 Kohlbrenner, E., Aslanidi, G., Nash, K., Shklyaev, S., Campbell-Thompson, M., Byrne, B. J., Snyder, R. O., Muzyczka, N., Warrington, K. H. and Zolotukhin, S. 2005. Successful production of pseudotyped rAAV vectors using a modified baculovirus expression system. Mol. Ther. 12, 1217-1225.
19 Kuck, D., Kern, A. and Kleinschmidt, J. A. 2007. Development of AAV serotype-specific ELISAs using novel monoclonal antibodies. J. Virol. Methods 140, 17-24.   DOI
20 Le, C. T., Gray, G. C. and Poddar, S. K. 2001. A modified rapid method of nucleic acid isolation from suspension of matured virus: applied in restriction analysis of DNA from an adenovirus prototype strain and a patient isolate. J. Med. Microbiol. 50, 571-574.
21 Mayginnes, J. P., Reed, S. E., Berg, H. G., Staley, E. M., Pintel, D. J. and Tullis, G. E. 2006. Quantitation of encapsidated recombinant adeno-associated virus DNA in crude cell lysates and tissue culture medium by quantitative, real-time PCR. J. Virol. Methods 137, 193-204.   DOI
22 Merten, O. W., Geny-Fiamma, C. and Douar, A. M. 2005. Current issues in adeno-associated viral vector production. Gene Ther. 12, S51-61.   DOI   ScienceOn
23 Park, J. Y., Lim, B. P., Lee, K., Kim, Y. G. and Jo, E. C. 2006. Scalable production of adeno-associated virus type 2 vectors via suspension transfection. Biotechnol. Bioeng. 20, 416-430.
24 Parks, W. P., Melnick, J. L., Rongey, R. and Mayor, H. D. 1967. Physical assay and growth cycle studies of a defective adeno-satellite virus. J. Virol. 1, 171-180.
25 Urabe, M., Ding, C. and Kotin, R. M. 2002. Insect cells as a factory to produce adeno-associated virus type 2 vectors. Hum. Gene Ther. 13, 1935-1943.   DOI
26 Qiao, C., Wang, B., Zhu, X., Li, J. and Xiao, X. 2002. A novel gene expression control system and its use in stable, high-titer 293 cell-based adeno-associated virus packing cell lines. J. Virol. 76, 13015-13027   DOI
27 Sommer, J. M., Smith, P. H., Parthasarathy, S., Isaacs, J., Vijay, S., Kieran, J., Powell, S. K., McClelland, A. and Wright, J. F. 2003. Quantification of adeno-associated virus particles and empty capsids by optical density measurement. Mol. Ther. 7, 122-128.   DOI
28 Transfiguracion, J., Jorio, H., Meghrous, J., Jacob, D. and Kamen, A. 2007. High yield purification of functional baculovirus vectors by size exclusion chromatography. J. Virol. Methods 142, 21-28.   DOI
29 Urabe, M., Nakakura, T., Xin, K. Q., Obara, Y., Mizukami, H., Kume, A., Kotin, R. M. and Ozawa, K. 2006. Scalable generation of high-titer recombinant adeno-associated virus type 5 in insect cells. J. Virol. 80, 1874-1885.   DOI
30 Veldwijk, M. R., Topaly, J., Laufs, S., Hengge, U. R., Wenz, F., Zeller, W. J. and Fruehauf, S. 2002. Development and optimization of a real-time quantitative PCR-based method for the titration of AAV-2 vector stocks. Mol. Ther. 6, 272-278.   DOI
31 Yun, H. J., Cho, Y. H., Moon, Y., Park, Y. W., Yoon, H. K., Kim, Y. J., Cho, S. H., Lee, Y. I., Kang, B. S., Kim, W. J., Park, K. and Seo, W. 2008. Transcriptional targeting of gene expression in breast cancer by the promoters of protein regulator of cytokinesis 1 and ribonuclease reductase 2. Exp. Mol. Med. 40, 345-353.   DOI   ScienceOn