Comparison of different technologies for producing recombinant adeno-associated virus on a laboratory scale

Adeno-associated virus vectors are among the most promising ones for the delivery of transgenes to various organs and tissues. Recombinant adeno-associated virus (rAAV) is able to transduce both dividing and non-dividing cells, has low immunogenicity, and is able to provide long-term expression of transgenes. Modern technologies make it possible to obtain rAAV for in vivo use, but they are not without drawbacks associated with laboriousness, scalability difficulties, and high cost, therefore, improvement of technological schemes for obtaining rAAV is an urgent issue. The aim of the study was to compare different technological approaches to rAAV production based on different conditions of the transfected HEK293 cell line cultivation on a laboratory scale. Materials and methods: HEK293 cell culture, AAV-DJ Packaging System, PlasmidSelect Xtra Starter Kit were used in the study. The technologies were compared using a model rAAV vector with a single-domain antibody transgene fused to the Fc-fragment of IgG1 specific to botulinum toxin. HEK293 cells were transfected with supercoiled plasmid DNA isolated by three-step chromatographic purification. The identity of the rAAV preparation was determined by electrophoresis, immunoblotting, and real-time polymerase chain reaction. Results: the study demonstrated the efficiency of the chromatographic method for obtaining a supercoiled form of plasmid DNA that can be used for efficient transfection of cell culture in order to produce rAAV. The study compared the following processes of rAAV production: using transient transfection and cultivation of the transfected HEK293 cell suspension in Erlenmeyer flasks, adherent culture in T-flasks, and adherent culture in a BioBLU 5p bioreactor on a matrix of Fibra-Cel disks. Conclusions: the data obtained showed the possibility of using the described approaches to purification of plasmid DNA, cell transfection, and cultivation of the transfected cells under various conditions to obtain rAAV samples that expresses the antibody gene. The BioBLU 5p reactor with Fibra-Cel discs was used for the first time to produce preparative quantities of rAAV on a laboratory scale, which increased the adherent surface area during cell culture and transfection, and, as a result, increased the yield of the target product.

1. Naso MF, Tomkowicz B, Perry WL, Strohl WR. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs. 2017;31(4):317–34. https://doi.org/10.1007/s40259-017-0234-5

2. Samulski RJ, Muzyczka N. AAV-mediated gene therapy for research and therapeutic purposes. Annu Rev Virol. 2014;1(1):427–51. https://doi.org/10.1146/annurev-virology-031413-085355

3. Choi VW, McCarty DM, Samulski RJ. Host cell DNA repair pathways in adeno-associated viral genome processing. J Virol. 2006;80(21):10346–56. https://doi.org/10.1128/JVI.00841-06

4. Mays LE, Wang L, Lin J, Bell P, Crawford A, Wherry EJ, Wilson JM. AAV8 induces tolerance in murine muscle as a result of poor APC transduction, T cell exhaustion, and minimal MHCI upregulation on target cells. Mol Ther. 2014;22(1):28–41. https://doi.org/10.1038/mt.2013.134

5. Ahi YS, Bangari DS, Mittal SK. Adenoviral vector immunity: its implications and circumvention strategies. Curr Gene Ther. 2011;11(4):307–20. https://doi.org/10.2174/156652311796150372

6. Grieger JC, Soltys SM, Samulski RJ. Production of recombinant adeno-associated virus vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX and FLT1 clinical vector. Mol Ther. 2016;24(2):287–97. https://doi.org/10.1038/mt.2015.187

7. Rajendra Y, Kiseljak D, Baldi L, Wurm FM, Hacker DL. Transcriptional and post-transcriptional limitations of high-yielding, PEI-mediated transient transfection with CHO and HEK-293E cells. Biotechnol Prog. 2015;31(2):541–9. https://doi.org/10.1002/btpr.2064

8. Wright JF. Transient transfection methods for clinical adeno-associated viral vector production. Hum Gene Ther. 2009;20(7):698–706. https://doi.org/10.1089/hum.2009.064

9. Keeler AM, Flotte TR. Recombinant adeno-associated virus gene therapy in light of Luxturna (and Zolgensma and Glybera): Where are we, and how did we get here? Annu Rev Virol. 2019;6(1):601–21. https://doi.org/10.1146/annurev-virology-092818-015530

10. Godakova SA, Noskov AN, Vinogradova ID, Ugriumova GA, Solovyev AI, Esmagambetov IB, et al. Camelid VHHs fused to human Fc fragments provide long term protection against botulinum neurotoxin A in mice. Toxins (Basel). 2019;11(8):464. https://doi.org/10.3390/toxins11080464

11. Froger A, Hall JE. Transformation of plasmid DNA into E. coli using the heat shock method. J Vis Exp. 2007;6:253. https://doi.org/10.3791/253

12. Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979;7(6):1513–23. https://doi.org/10.1093/nar/7.6.1513

13. Merten OW. AAV vector production: state of the art developments and remaining challenges. Cell Gene Therapy Insights. 2016;2(5):521–51.

14. Sousa F, Prazeres DMF, Queiroz JA. Improvement of transfection efficiency by using supercoiled plasmid DNA purified with arginine affinity chromatography. J Gene Med. 2009;11(1):79–88. https://doi.org/10.1002/jgm.1272

15. Diogo MM, Queiroz JA, Prazeres DMF. Chromatography of plasmid DNA. J Chromatogr A. 2005;1069(1):3–22. https://doi.org/10.1016/j.chroma.2004.09.050

16. Stadler J, Lemmens R, Nyhammar T. Plasmid DNA purification. J Gene Med. 2004;6(Suppl 1):S54–S66. https://doi.org/10.1002/jgm.512

17. Cupillard L, Juillard V, Latour S, Colombet G, Cachet N, Richard S, et al. Impact of plasmid supercoiling on the efficacy of a rabies DNA vaccine to protect cats. Vaccine. 2005;23(16):1910–6. https://doi.org/10.1016/j.vaccine.2004.10.018

18. Zhao H, Lee KJ, Daris M, Lin Y, Wolfe T, Sheng J, et al. Creation of a high-yield AAV vector production platform in suspension cells using a design of experiment approach. Mol Ther Methods Clin Dev. 2020;18:312–20. https://doi.org/10.1016/j.omtm.2020.06.004

19. Nass SA, Mattingly MA, Woodcock DA, Burnham BL, Ardinger JA, Osmond SE, et al. Universal method for the purification of recombinant AAV vectors of differing serotypes. Mol Ther Methods Clin Dev. 2017;9:33–46. https://doi.org/10.1016/j.omtm.2017.12.004

20. Sommer JM, Smith PH, Parthasarathy S, Isaacs J, Vijay S, Kieran J, et al. Quantification of adeno-associated virus particles and empty capsids by optical density measurement. Mol Ther. 2003;7(1):122–8. https://doi.org/10.1016/s1525-0016(02)00019-9

21. Benskey MJ, Sandoval IM, Manfredsson FP. Continuous Collection of Adeno-Associated Virus from Producer Cell Medium Significantly Increases Total Viral Yield. Hum Gene Ther Methods. 2016;27(1):32–45. https://doi.org/10.1089/hgtb.2015.117

22. Hajba L, Guttman A. Recent Advances in the Analysis Full/Empty Capsid Ratio and Genome Integrity of Adeno-associated Virus (AAV) Gene Delivery Vectors. Curr Mol Med. 2020;20(10):806–13. https://doi.org/10.2174/1566524020999200730181042

23. Crosson SM, Dib P, Smith KJ, Zolotukhin S. Helper-free production of laboratory grade AAV and purification by iodixanol density gradient centrifugation. Mol Ther Methods Clin Dev. 2018;10:1–7. https://doi.org/10.1016/j.omtm.2018.05.001