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Characterization of Viral Genome Encapsidated in Adeno-associated Recombinant Vectors Produced in Yeast Saccharomyces cerevisiae

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Abstract

Adeno-associated virus (AAV) is a small, non-enveloped virus used as vector in gene therapy, mainly produced in human cells and in baculovirus systems. Intense studies on these platforms led to the production of vectors with titers between 103 and 105 viral genomes (vg) per cells. In spite of this, vector yields need to be improved to satisfy the high product demands of clinical trials and future commercialization. Our studies and those of other groups have explored the possibility to exploit the yeast Saccharomyces cerevisiae to produce rAAV. We previously demonstrated that yeast supports AAV genome replication and capsid assembly. The purpose of this study was to evaluate the quality of the encapsidated AAV DNA. Here, we report the construction of a yeast strain expressing Rep68/40 from an integrated copy of the Rep gene under the control of the yeast constitutive ADH promoter and Capsid proteins from the Cap gene under the control of an inducible GAL promoter. Our results indicate that a portion of AAV particles generated by this system contains encapsidated AAV DNA. However, the majority of encapsidated DNA consists of fragmented regions of the transgene cassette, with ITRs being the most represented sequences. Altogether, these data indicate that, in yeast, encapsidation occurs with low efficiency and that rAAVs resemble pseudo-vectors that are present in clinical-grade rAAV preparations.

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References

  1. Mendell, J. R., Al-Zaidy, S., Shell, R., Arnold, W. D., Rodino-Klapac, L. R., Prior, T. W., et al. (2017). Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy. New England Journal of Medicine, 377, 1713–1722.

    CAS  Google Scholar 

  2. Le Meur, G., Lebranchu, P., Billaud, F., Adjali, O., Schmitt, S., Bezieau, S., et al. (2018). Safety and Long-Term Efficacy of AAV4 Gene Therapy in Patients with RPE65 Leber Congenital Amaurosis. Molecular Therapy, 26, 256–268.

    PubMed  Google Scholar 

  3. Kassner, U., Hollstein, T., Grenkowitz, T., Wuhle-Demuth, M., Salewsky, B., Demuth, I., et al. (2018). Gene therapy in lipoprotein lipase deficiency: case report on the first patient treated with alipogene tiparvovec under daily practice conditions. Human Gene Therapy, 29, 520–527.

    CAS  PubMed  Google Scholar 

  4. Li, C., & Samulski, R. J. (2020). Engineering adeno-associated virus vectors for gene therapy. Nature Reviews Genetics, 21, 255–272.

    CAS  PubMed  Google Scholar 

  5. Hermonat, P. L., & Muzyczka, N. (1984). Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture cells. Proc Natl Acad Sci U S A, 81, 6466–6470.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Galibert, L., & Merten, O. W. (2011). Latest developments in the large-scale production of adeno-associated virus vectors in insect cells toward the treatment of neuromuscular diseases. Journal of Invertebrate Pathology, 107(Suppl), S80-93.

    CAS  PubMed  Google Scholar 

  7. Penaud-Budloo, M., Francois, A., Clement, N., & Ayuso, E. (2018). Pharmacology of recombinant Adeno-associated virus production. Mol Ther Methods Clin Dev, 8, 166–180.

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Mietzsch, M., Grasse, S., Zurawski, C., Weger, S., Bennett, A., Agbandje-McKenna, M., et al. (2014). OneBac: platform for scalable and high-titer production of adeno-associated virus serotype 1–12 vectors for gene therapy. Human Gene Therapy, 25, 212–222.

    CAS  PubMed  Google Scholar 

  9. Kim, H. J. (2017). Yeast as an expression system for producing virus-like particles: what factors do we need to consider? Letters in Applied Microbiology, 64, 111–123.

    CAS  PubMed  Google Scholar 

  10. Zhao, R. Y. (2017). Yeast for virus research. Microb. Cell, 4, 311–330.

    CAS  Google Scholar 

  11. Barajas, D., Aponte-Ubillus, J. J., Akeefe, H., Cinek, T., Peltier, J., & Gold, D. (2017). Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae. PLoS ONE, 12, e0173010.

    PubMed  PubMed Central  Google Scholar 

  12. Cervelli, T., Backovic, A., & Galli, A. (2011). Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae. PLoS ONE, 6, e23474.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Backovic, A., Cervelli, T., Salvetti, A., Zentilin, L., Giacca, M., & Galli, A. (2012). Capsid protein expression and adeno-associated virus like particles assembly in Saccharomyces cerevisiae. Microbial Cell Factories, 11, 124.

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Galli, A., Della Latta, V., Bologna, C., Pucciarelli, D., Cipriani, F., Backovic, A., et al. (2017). Strategies to optimize capsid protein expression and single-stranded DNA formation of adeno-associated virus in Saccharomyces cerevisiae. Journal of Applied Microbiology, 123, 414–428.

    CAS  PubMed  Google Scholar 

  15. Aponte-Ubillus, J. J., Barajas, D., Sterling, H., Aghajanirefah, A., Bardliving, C., Peltier, J., et al. (2020). Proteome profiling and vector yield optimization in a recombinant adeno-associated virus-producing yeast model. Microbiologyopen. https://doi.org/10.1002/mbo3.1136.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Ling, C., Wang, Y., Lu, Y., Wang, L., Jayandharan, G. R., Aslanidi, G. V., et al. (2015). The Adeno-Associated Virus Genome Packaging Puzzle. J Mol Genet Med 9.

  17. Grimm, D., Kern, A., Pawlita, M., Ferrari, F., Samulski, R., & Kleinschmidt, J. (1999). Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2. Gene Therapy, 6, 1322–1330.

    CAS  PubMed  Google Scholar 

  18. King, J. A., Dubielzig, R., Grimm, D., & Kleinschmidt, J. A. (2001). DNA helicase-mediated packaging of adeno-associated virus type 2 genomes into preformed capsids. EMBO Journal, 20, 3282–3291.

    CAS  Google Scholar 

  19. Chadeuf, G., Ciron, C., Moullier, P., & Salvetti, A. (2005). Evidence for encapsidation of prokaryotic sequences during recombinant adeno-associated virus production and their in vivo persistence after vector delivery. Molecular Therapy, 12, 744–753.

    CAS  PubMed  Google Scholar 

  20. Kapranov, P., Chen, L., Dederich, D., Dong, B., He, J., Steinmann, K. E., et al. (2012). Native molecular state of adeno-associated viral vectors revealed by single-molecule sequencing. Human Gene Therapy, 23, 46–55.

    CAS  PubMed  Google Scholar 

  21. Nathwani, A. C., Tuddenham, E. G., Rangarajan, S., Rosales, C., McIntosh, J., Linch, D. C., et al. (2011). Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. New England Journal of Medicine, 365, 2357–2365.

    CAS  Google Scholar 

  22. de la Maza, L. M., & Carter, B. J. (1980). Molecular structure of adeno-associated virus variant DNA. Journal of Biological Chemistry, 255, 3194–3203.

    Google Scholar 

  23. Senapathy, P., & Carter, B. J. (1984). Molecular cloning of adeno-associated virus variant genomes and generation of infectious virus by recombination in mammalian cells. Journal of Biological Chemistry, 259, 4661–4666.

    CAS  Google Scholar 

  24. Wang, Q., Dong, B., Pokiniewski, K. A., Firrman, J., Wu, Z., Chin, M. P., et al. (2017). Syngeneic AAV Pseudo-particles Potentiate Gene Transduction of AAV Vectors. Mol Ther Methods Clin Dev, 4, 149–158.

    CAS  PubMed  Google Scholar 

  25. Gietz, R. D., & Schiestl, R. H. (2007). Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols, 2, 38–41.

    CAS  PubMed  Google Scholar 

  26. Oldenburg, K. R., Vo, K. T., Michaelis, S., & Paddon, C. (1997). Recombination-mediated PCR-directed plasmid construction in vivo in yeast. Nucleic Acids Research, 25, 451–452.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Deodato, B., Arsic, N., Zentilin, L., Galeano, M., Santoro, D., Torre, V., et al. (2002). Recombinant AAV vector encoding human VEGF165 enhances wound healing. Gene Therapy, 9, 777–785.

    CAS  PubMed  Google Scholar 

  28. Arsic, N., Zacchigna, S., Zentilin, L., Ramirez-Correa, G., Pattarini, L., Salvi, A., et al. (2004). Vascular endothelial growth factor stimulates skeletal muscle regeneration in vivo. Molecular Therapy, 10, 844–854.

    CAS  PubMed  Google Scholar 

  29. Zentilin, L., Marcello, A., & Giacca, M. (2001). Involvement of cellular double-stranded DNA break binding proteins in processing of the recombinant adeno-associated virus genome. Journal of Virology, 75, 12279–12287.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Trapani, I., Toriello, E., de Simone, S., Colella, P., Iodice, C., Polishchuk, E. V., et al. (2015). Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease. Human Molecular Genetics, 24, 6811–6825.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Aurnhammer, C., Haase, M., Muether, N., Hausl, M., Rauschhuber, C., Huber, I., et al. (2012). Universal real-time PCR for the detection and quantification of adeno-associated virus serotype 2-derived inverted terminal repeat sequences. Hum Gene Ther Methods, 23, 18–28.

    CAS  PubMed  Google Scholar 

  32. Maurer, A. C., & Weitzman, M. D. (2020). Adeno-Associated Virus Genome Interactions Important for Vector Production and Transduction. Human Gene Therapy, 31, 499–511.

    CAS  PubMed  Google Scholar 

  33. Dubielzig, R., King, J. A., Weger, S., Kern, A., & Kleinschmidt, J. A. (1999). Adeno-associated virus type 2 protein interactions: formation of pre-encapsidation complexes. Journal of Virology, 73, 8989–8998.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Owens, R. A., Weitzman, M. D., Kyostio, S. R., & Carter, B. J. (1993). Identification of a DNA-binding domain in the amino terminus of adeno-associated virus Rep proteins. Journal of Virology, 67, 997–1005.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Im, D. S., & Muzyczka, N. (1990). The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell, 61, 447–457.

    CAS  PubMed  Google Scholar 

  36. Prasad, K. M., & Trempe, J. P. (1995). The adeno-associated virus Rep78 protein is covalently linked to viral DNA in a preformed virion. Virology, 214, 360–370.

    CAS  PubMed  Google Scholar 

  37. Myers, M. W., & Carter, B. J. (1981). Adeno-associated virus replication. The effect of L-canavanine or a helper virus mutation on accumulation of viral capsids and progeny single-stranded DNA. Journal of Biological Chemistry, 256, 567–570.

    CAS  Google Scholar 

  38. McCarty, D. M., Pereira, D. J., Zolotukhin, I., Zhou, X., Ryan, J. H., & Muzyczka, N. (1994). Identification of linear DNA sequences that specifically bind the adeno-associated virus Rep protein. Journal of Virology, 68, 4988–4997.

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Nony, P., Tessier, J., Chadeuf, G., Ward, P., Giraud, A., Dugast, M., et al. (2001). Novel cis-acting replication element in the adeno-associated virus type 2 genome is involved in amplification of integrated rep-cap sequences. Journal of Virology, 75, 9991–9994.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Nony, P., Chadeuf, G., Tessier, J., Moullier, P., & Salvetti, A. (2003). Evidence for packaging of rep-cap sequences into adeno-associated virus (AAV) type 2 capsids in the absence of inverted terminal repeats: a model for generation of rep-positive AAV particles. Journal of Virology, 77, 776–781.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Yoon-Robarts, M., Blouin, A. G., Bleker, S., Kleinschmidt, J. A., Aggarwal, A. K., Escalante, C. R., et al. (2004). Residues within the B’ motif are critical for DNA binding by the superfamily 3 helicase Rep40 of adeno-associated virus type 2. Journal of Biological Chemistry, 279, 50472–50481.

    CAS  Google Scholar 

  42. Dong, B., Nakai, H., & Xiao, W. (2010). Characterization of genome integrity for oversized recombinant AAV vector. Molecular Therapy, 18, 87–92.

    CAS  PubMed  Google Scholar 

  43. Wu, Z., Yang, H., & Colosi, P. (2010). Effect of genome size on AAV vector packaging. Molecular Therapy, 18, 80–86.

    CAS  PubMed  Google Scholar 

  44. D’Costa, S., Blouin, V., Broucque, F., Penaud-Budloo, M., Francois, A., Perez, I. C., et al. (2016). Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR. Mol Ther Methods Clin Dev, 5, 16019.

    PubMed  PubMed Central  Google Scholar 

  45. Werling, N. J., Satkunanathan, S., Thorpe, R., & Zhao, Y. (2015). Systematic Comparison and Validation of Quantitative Real-Time PCR Methods for the Quantitation of Adeno-Associated Viral Products. Hum Gene Ther Methods, 26, 82–92.

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Dobnik, D., Kogovsek, P., Jakomin, T., Kosir, N., Tusek Znidaric, M., Leskovec, M., et al. (2019). Accurate Quantification and Characterization of Adeno-Associated Viral Vectors. Front Microbiol, 10, 1570.

    PubMed  PubMed Central  Google Scholar 

  47. Berns, K. I., & Rose, J. A. (1970). Evidence for a single-stranded adenovirus-associated virus genome: isolation and separation of complementary single strands. Journal of Virology, 5, 693–699.

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Zhou, X., Zeng, X., Fan, Z., Li, C., McCown, T., Samulski, R. J., et al. (2008). Adeno-associated virus of a single-polarity DNA genome is capable of transduction in vivo. Molecular Therapy, 16, 494–499.

    CAS  PubMed  Google Scholar 

  49. McAlister, V. J., & Owens, R. A. (2010). Substitution of adeno-associated virus Rep protein binding and nicking sites with human chromosome 19 sequences. Virol J, 7, 218.

    PubMed  PubMed Central  Google Scholar 

  50. Qu, G., Bahr-Davidson, J., Prado, J., Tai, A., Cataniag, F., McDonnell, J., et al. (2007). Separation of adeno-associated virus type 2 empty particles from genome containing vectors by anion-exchange column chromatography. Journal of Virological Methods, 140, 183–192.

    CAS  PubMed  Google Scholar 

  51. Samulski, R. J., & Muzyczka, N. (2014). AAV-Mediated Gene Therapy for Research and Therapeutic Purposes. Annu Rev Virol, 1, 427–451.

    PubMed  Google Scholar 

  52. Aponte-Ubillus, J. J., Barajas, D., Peltier, J., Bardliving, C., Shamlou, P., & Gold, D. (2018). Molecular design for recombinant adeno-associated virus (rAAV) vector production. Applied Microbiology and Biotechnology, 102, 1045–1054.

    CAS  PubMed  Google Scholar 

  53. Aponte-Ubillus, J. J., Barajas, D., Peltier, J., Bardliving, C., Shamlou, P., & Gold, D. (2019). A rAAV2-producing yeast screening model to identify host proteins enhancing rAAV DNA replication and vector yield. Biotechnology Progress, 35, e2725.

    PubMed  Google Scholar 

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Acknowledgements

This work was supported by the Grant 127/16 funded by the “Fondazione Pisa” assigned to AG. We thank Dr Milena Rizzo for helpful suggestions on qPCR. We thanks also Marina Dapas and Michela Zotti of the AVU (AAV vector unit) core facility of ICGEB, Trieste, for AAV production in human cells.

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Authors and Affiliations

  1. Yeast Genetics and Genomics Group, Institute of Clinical Physiology, CNR, via Moruzzi 1, 56124, Pisa, Italy

    Alvaro Galli, Ilenia Iaia, Maria Serena Milella, Filippo Cipriani, Veronica Della Latta & Tiziana Cervelli

  2. School of Cardiovascular Medicine & Sciences, British Heart Foundation Centre of Research Excellence, King’s College London, SE5 9NU London, United Kingdom

    Mauro Giacca

  3. Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy

    Mauro Giacca & Lorena Zentilin

  4. Department of Medical, Surgical and Health Sciences, University of Trieste, Trieste, Italy

    Mauro Giacca

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  1. Alvaro Galli
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  2. Ilenia Iaia
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  3. Maria Serena Milella
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  4. Filippo Cipriani
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  5. Veronica Della Latta
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  8. Tiziana Cervelli
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Contributions

AG and TC designed the study. LZ produced rAAV from human cells. II, SM, FC and VD performed the study. AG, II, SM, FC, VD and TC analysed the results. LZ, MG, AG and TC discussed the results. AG and TC wrote the manuscript. All authors revised the manuscript and agreed to be accountable for all aspects of the presented work.

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Correspondence to Tiziana Cervelli.

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Galli, A., Iaia, I., Milella, M.S. et al. Characterization of Viral Genome Encapsidated in Adeno-associated Recombinant Vectors Produced in Yeast Saccharomyces cerevisiae. Mol Biotechnol 63, 156–165 (2021). https://doi.org/10.1007/s12033-020-00294-4

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