Inducible HEK293 AAV Packaging Cell Lines Expressing Rep Proteins

Overview

Journal Mol Ther Methods Clin Dev

Publisher Cell Press

Date 2023 Aug 10

PMID 37560197

Authors

Lovro Jalsic

Viktoria Lytvyn

Seyyed Mehdy Elahi

Sabahudin Hrapovic

Nasha Nassoury

Parminder Singh Chahal

Bruno Gaillet

Renald Gilbert

Affiliations

Soon will be listed here.

Abstract

Packaging or producer cell lines for scalable recombinant adeno-associated virus (rAAV) production have been notoriously difficult to create due in part to the cytostatic nature of the Rep proteins required for AAV production. The most difficult challenge being creating AAV packaging cell lines using HEK293 parental cells, currently the best mammalian platform for rAAV production due to the constitutive expression of in HEK293 cells, a key transcription activator. Using suspension and serum-free media adapted HEK293SF carrying a gene expression regulation system induced by addition of cumate and coumermycin, we were able to create -expressing AAV packaging cells. This was achieved by carefully choosing two of the AAV Rep proteins (Rep 40 and 68), using two inducible promoters with different expression levels and integrating into the cells through lentiviral vector transduction. Three of our best clones produced rAAV titers comparable to titers obtained by standard triple plasmid transfection of their parental cells. These clones were stable for up to 7 weeks under continuous cultures condition. rAAV production from one clone was also validated at scale of 1 L in a wave bioreactor using serum-free suspension culture.

Citing Articles

Polo-like kinase inhibitors increase AAV production by halting cell cycle progression.

Fisher K, Grafton F, Ispaso F, Tworig J, Derler R, Sonntag F Mol Ther Methods Clin Dev. 2025; 33(1):101412.

PMID: 39968185 PMC: 11834096. DOI: 10.1016/j.omtm.2025.101412.

Enhanced ER protein processing gene expression increases rAAV yield and full capsid ratio in HEK293 cells.

Fu Q, Wang Y, Qin J, Xie D, McNally D, Yoon S Appl Microbiol Biotechnol. 2024; 108(1):459.

PMID: 39230729 PMC: 11374875. DOI: 10.1007/s00253-024-13281-5.

Therapeutic Application and Structural Features of Adeno-Associated Virus Vector.

Matsuzaka Y, Yashiro R Curr Issues Mol Biol. 2024; 46(8):8464-8498.

PMID: 39194716 PMC: 11353222. DOI: 10.3390/cimb46080499.

Adeno-associated virus as a delivery vector for gene therapy of human diseases.

Wang J, Gessler D, Zhan W, Gallagher T, Gao G Signal Transduct Target Ther. 2024; 9(1):78.

PMID: 38565561 PMC: 10987683. DOI: 10.1038/s41392-024-01780-w.

Advances of Recombinant Adenoviral Vectors in Preclinical and Clinical Applications.

Scarsella L, Ehrke-Schulz E, Paulussen M, Thal S, Ehrhardt A, Aydin M Viruses. 2024; 16(3).

PMID: 38543743 PMC: 10974029. DOI: 10.3390/v16030377.

References

Chang L, Shi Y, Shenk T . Adeno-associated virus P5 promoter contains an adenovirus E1A-inducible element and a binding site for the major late transcription factor. J Virol. 1989; 63(8):3479-88. PMC: 250925. DOI: 10.1128/JVI.63.8.3479-3488.1989. View

James J, Aggarwal A, Linden R, Escalante C . Structure of adeno-associated virus type 2 Rep40-ADP complex: insight into nucleotide recognition and catalysis by superfamily 3 helicases. Proc Natl Acad Sci U S A. 2004; 101(34):12455-60. PMC: 515083. DOI: 10.1073/pnas.0403454101. View

Broussau S, Jabbour N, Lachapelle G, Durocher Y, Tom R, Transfiguracion J . Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture. Mol Ther. 2008; 16(3):500-7. DOI: 10.1038/sj.mt.6300383. View

Li J, Samulski R, Xiao X . Role for highly regulated rep gene expression in adeno-associated virus vector production. J Virol. 1997; 71(7):5236-43. PMC: 191759. DOI: 10.1128/JVI.71.7.5236-5243.1997. View

Inoue N, Russell D . Packaging cells based on inducible gene amplification for the production of adeno-associated virus vectors. J Virol. 1998; 72(9):7024-31. PMC: 109922. DOI: 10.1128/JVI.72.9.7024-7031.1998. View

Prasad K, Zhou C, Trempe J . Characterization of the Rep78/adeno-associated virus complex. Virology. 1997; 229(1):183-92. DOI: 10.1006/viro.1996.8431. View

Rommelaere J, Cornelis J . Antineoplastic activity of parvoviruses. J Virol Methods. 1991; 33(3):233-51. DOI: 10.1016/0166-0934(91)90024-t. View

Grimm D, Lee J, Wang L, Desai T, Akache B, Storm T . In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses. J Virol. 2008; 82(12):5887-911. PMC: 2395137. DOI: 10.1128/JVI.00254-08. View

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(16):4351-61. PMC: 302033. DOI: 10.1093/emboj/19.16.4351. View

10.

Mizukami H, Okada T, Ogasawara Y, Matsushita T, Urabe M, Kume A . Separate control of Rep and Cap expression using mutant and wild-type loxP sequences and improved packaging system for adeno-associated virus vector production. Mol Biotechnol. 2004; 27(1):7-14. DOI: 10.1385/MB:27:1:07. View

11.

Chadeuf G, Favre D, Tessier J, Provost N, Nony P, Kleinschmidt J . 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(4):260-8. DOI: 10.1002/1521-2254(200007/08)2:4<260::AID-JGM111>3.0.CO;2-8. View

12.

Davis M, Wu J, Owens R . Mutational analysis of adeno-associated virus type 2 Rep68 protein endonuclease activity on partially single-stranded substrates. J Virol. 2000; 74(6):2936-42. PMC: 111789. DOI: 10.1128/jvi.74.6.2936-2942.2000. View

13.

Holscher C, Kleinschmidt J, Burkle A . High-level expression of adeno-associated virus (AAV) Rep78 or Rep68 protein is sufficient for infectious-particle formation by a rep-negative AAV mutant. J Virol. 1995; 69(11):6880-5. PMC: 189603. DOI: 10.1128/JVI.69.11.6880-6885.1995. View

14.

Thorne B, Takeya R, Peluso R . Manufacturing recombinant adeno-associated viral vectors from producer cell clones. Hum Gene Ther. 2009; 20(7):707-14. DOI: 10.1089/hum.2009.070. View

15.

Qiao C, Li J, Skold A, Zhang X, Xiao X . Feasibility of generating adeno-associated virus packaging cell lines containing inducible adenovirus helper genes. J Virol. 2002; 76(4):1904-13. PMC: 135905. DOI: 10.1128/jvi.76.4.1904-1913.2002. View

16.

Louis N, Evelegh C, Graham F . Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line. Virology. 1997; 233(2):423-9. DOI: 10.1006/viro.1997.8597. View

17.

Horer M, Weger S, Butz K, Hoppe-Seyler F, Geisen C, Kleinschmidt J . Mutational analysis of adeno-associated virus Rep protein-mediated inhibition of heterologous and homologous promoters. J Virol. 1995; 69(9):5485-96. PMC: 189398. DOI: 10.1128/JVI.69.9.5485-5496.1995. View

18.

Nakamura S, Nakamura R, Shibata K, Kobayashi M, Sahara N, Shigeno K . Development of packaging cell lines for generation of adeno-associated virus vectors by lentiviral gene transfer of trans-complementary components. Eur J Haematol. 2004; 73(4):285-94. DOI: 10.1111/j.1600-0609.2004.00290.x. View

19.

Aurnhammer C, Haase M, Muether N, Hausl M, Rauschhuber C, Huber I . Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum Gene Ther Methods. 2012; 23(1):18-28. DOI: 10.1089/hgtb.2011.034. View

20.

Gao G, Qu G, Faust L, Engdahl R, Xiao W, Hughes J . High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus. Hum Gene Ther. 1998; 9(16):2353-62. DOI: 10.1089/hum.1998.9.16-2353. View