Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Rapid characterization of adeno-associated virus (AAV) gene therapy vectors by mass photometry

Gene Therapy volume 29, pages 691–697 (2022)Cite this article

Subjects

Abstract

Recombinant adeno-associated viruses (rAAV) are used extensively as gene delivery vectors in clinical studies, and several rAAV based treatments have already been approved. Significant progress has been made in rAAV manufacturing; however, better and more precise capsid characterization techniques are still needed to guarantee the purity and safety of rAAV preparations. Current analytical techniques used to characterize rAAV preparations are susceptible to background signals, have limited accuracy, or require a large amount of time and material. A recently developed single-molecule technique, mass photometry (MP), measures mass distributions of biomolecules with high-resolution and sensitivity. Here we explore applications of MP for the characterization of capsid fractions. We demonstrate that MP is able to resolve and quantify not only empty and full-genome containing capsid populations but also identify partially packaged capsid impurities. MP data accurately measures full and empty capsid ratios, and can be used to estimate the size of the encapsidated genome. MP distributions provide information on sample heterogeneity and on the presence of aggregates. Sub-picomole quantities of sample are sufficient for MP analysis, and data can be obtained and analyzed within minutes. This method provides a simple, robust, and effective tool to monitor the physical attributes of rAAV vectors.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: KDE mass distributions of the purified rAAV preparations.
Fig. 2: Replicates of the MP measurements of the empty rAAV capsids.
Fig. 3: MP determination of the full to empty capsid ratios.
Fig. 4: MP distributions peak positions plotted against the sample genome size.
Fig. 5: Comparison of the SV-AUC and MP data.
Fig. 6: Replicates of the MP measurements of the scAAV5-CMV-GFP vectors.

References

  1. Kotin RM, Siniscalco M, Samulski RJ, Zhu XD, Hunter L, Laughlin CA, et al. Site-specific integration by adeno-associated virus. Proc Natl Acad Sci U S A. 1990;87:2211–5.

    Article  CAS  Google Scholar 

  2. Surosky RT, Urabe M, Godwin SG, McQuiston SA, Kurtzman GJ, Ozawa K, et al. Adeno-associated virus Rep proteins target DNA sequences to a unique locus in the human genome. J Virol. 1997;71:7951–9.

    Article  CAS  Google Scholar 

  3. Mendell JR, Al-Zaidy SA, Rodino-Klapac LR, Goodspeed K, Gray SJ, Kay CN, et al. Current clinical applications of in vivo gene therapy with AAVs. Mol Ther. 2021;29:464–88.

    Article  CAS  Google Scholar 

  4. Bastola P, Song L, Gilger BC, Hirsch ML. Adeno-associated virus mediated gene therapy for corneal diseases. Pharmaceutics 2020;12:767.

  5. Fu YW, Dai XY, Wang WT, Yang ZX, Zhao JJ, Zhang JP, et al. Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing. Nucleic Acids Res. 2021;49:969–85.

    Article  CAS  Google Scholar 

  6. Hana S, Peterson M, McLaughlin H, Marshall E, Fabian AJ, McKissick O, et al. Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice. Gene Ther 2021;28:646–58. https://doi.org/10.1038/s41434-021-00224-2.

    Article  CAS  Google Scholar 

  7. Sonntag F, Schmidt K, Kleinschmidt JA. A viral assembly factor promotes AAV2 capsid formation in the nucleolus. Proc Natl Acad Sci U S A. 2010;107:10220–5.

    Article  CAS  Google Scholar 

  8. Srivastava A, Lusby EW, Berns KI. Nucleotide sequence and organization of the adeno-associated virus 2 genome. J Virol. 1983;45:555–64.

    Article  CAS  Google Scholar 

  9. Trempe JP, Carter BJ. Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein. J Virol. 1988;62:3356–63.

    Article  CAS  Google Scholar 

  10. Xie Q, Bu W, Bhatia S, Hare J, Somasundaram T, Azzi A. et al. The atomic structure of adeno-associated virus (AAV-2), a vector for human gene therapy. Proc Natl Acad Sci U S A. 2002;99:10405–10.

    Article  CAS  Google Scholar 

  11. de la Maza LM, Carter BJ. Molecular structure of adeno-associated virus variant DNA. J Biol Chem. 1980;255:3194–203.

    Article  Google Scholar 

  12. Lipps BV, Mayor HD. Characterization of heavy particles of adeno-associated virus type 1. J Gen Virol. 1982;58:63–72.

    Article  CAS  Google Scholar 

  13. Lock M, Alvira MR, Wilson JM. Analysis of particle content of recombinant adeno-associated virus serotype 8 vectors by ion-exchange chromatography. Hum Gene Ther Methods. 2012;23:56–64.

    Article  CAS  Google Scholar 

  14. Gao K, Li M, Zhong L, Su Q, Li J, Li S, et al. Empty virions in AAV8 vector preparations reduce transduction efficiency and may cause total viral particle dose-limiting side-effects. Mol Ther Methods Clin Dev. 2014;1:20139.

    Article  Google Scholar 

  15. Wright JF. AAV empty capsids: for better or for worse? Mol Ther. 2014;22:1–2.

    Article  CAS  Google Scholar 

  16. Gimpel AL, Katsidis G, Sha S, Maloney AJ, Hong MS, Nguyen TTN, et al. Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies. Molecular Therapy. 2021;20:740–54.

    CAS  Google Scholar 

  17. Dobnik D, Kogovsek P, Jakomin T, Kosir N, Tusek Znidaric M, Leskovec M, et al. Accurate quantification and characterization of adeno-associated viral vectors. Front Microbiol. 2019;10:1570.

    Article  Google Scholar 

  18. Kuck D, Kern A, Kleinschmidt JA. Development of AAV serotype-specific ELISAs using novel monoclonal antibodies. J Virol Methods. 2007;140:17–24.

    Article  CAS  Google Scholar 

  19. 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;7:122–8.

    Article  CAS  Google Scholar 

  20. McIntosh NL, Berguig GY, Karim OA, Cortesio CL, De Angelis R, Khan AA, et al. Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering. Sci Rep. 2021;11:3012.

    Article  CAS  Google Scholar 

  21. Subramanian S, Maurer AC, Bator CM, Makhov AM, Conway JF, Turner KB, et al. Filling adeno-associated virus capsids: estimating success by cryo-electron microscopy. Hum Gene Ther. 2019;30:1449–60.

    Article  CAS  Google Scholar 

  22. Urabe M, Xin KQ, Obara Y, Nakakura T, Mizukami H, Kume A, et al. Removal of empty capsids from type 1 adeno-associated virus vector stocks by anion-exchange chromatography potentiates transgene expression. Mol Ther. 2006;13:823–8.

    Article  CAS  Google Scholar 

  23. Pierson EE, Keifer DZ, Asokan A, Jarrold MF. Resolving adeno-associated viral particle diversity with charge detection mass spectrometry. Anal Chem. 2016;88:6718–25.

    Article  CAS  Google Scholar 

  24. Burnham B, Nass S, Kong E, Mattingly M, Woodcock D, Song A, et al. Analytical ultracentrifugation as an approach to characterize recombinant adeno-associated viral vectors. Hum Gene Ther Methods. 2015;26:228–42.

    Article  CAS  Google Scholar 

  25. Cole D, Young G, Weigel A, Sebesta A, Kukura P. Label-free single-molecule imaging with numerical-aperture-shaped interferometric scattering microscopy. ACS Photonics. 2017;4:211–6.

    Article  CAS  Google Scholar 

  26. Young G, Hundt N, Cole D, Fineberg A, Andrecka J, Tyler A, et al. Quantitative mass imaging of single biological macromolecules. Science. 2018;360:423–7.

    Article  CAS  Google Scholar 

  27. Li Y, Struwe WB, Kukura P. Single molecule mass photometry of nucleic acids. Nucleic Acids Res. 2020;48:e97.

    Article  CAS  Google Scholar 

  28. Olerinyova A, Sonn-Segev A, Gault J, Eichmann C, Schimpf J, Kopf AH, et al. Mass Photometry of Membrane Proteins Chem. 2021;7:2.

  29. Wu D, Piszczek G. Measuring the affinity of protein-protein interactions on a single-molecule level by mass photometry. Anal Biochem. 2020;592:113575.

    Article  CAS  Google Scholar 

  30. Hickman AB, Kailasan S, Genzor P, Haase AD, Dyda F. Casposase structure and the mechanistic link between DNA transposition and spacer acquisition by CRISPR-Cas. Elife 2020;9:e50004.

  31. Garmann RF, Goldfain AM, Manoharan VN. Measurements of the self-assembly kinetics of individual viral capsids around their RNA genome. Proc Natl Acad Sci U S A. 2019;116:22485–90.

    Article  CAS  Google Scholar 

  32. Malay AD, Miyazaki N, Biela A, Chakraborti S, Majsterkiewicz K, Stupka I, et al. An ultra-stable gold-coordinated protein cage displaying reversible assembly. Nature. 2019;569:438–42.

    Article  CAS  Google Scholar 

  33. Sonn-Segev A, Belacic K, Bodrug T, Young G, VanderLinden RT, Schulman BA, et al. Quantifying the heterogeneity of macromolecular machines by mass photometry. Nat Commun. 2020;11:1772.

    Article  CAS  Google Scholar 

  34. Yu W, Mookherjee S, Chaitankar V, Hiriyanna S, Kim JW, Brooks M, et al. Nrl knockdown by AAV-delivered CRISPR/Cas9 prevents retinal degeneration in mice. Nat Commun. 2017;8:14716.

    Article  Google Scholar 

  35. Wu D, Piszczek G. Standard protocol for mass photometry experiments. Eur Biophys J. 2021;50:403–9.

    Article  CAS  Google Scholar 

  36. Wu D, Piszczek G. Rapid determination of antibody-antigen affinity by mass photometry. JoVE. 2021. https://doi.org/10.3791/61784.

    Article  Google Scholar 

  37. Schuck P. Sedimentation velocity analytical ultracentrifugation: discrete species and size-distributions of macromolecules and particles, CRC Press: Boca Raton, FL; 2016.

  38. Berkowitz SA, Philo JS. Monitoring the homogeneity of adenovirus preparations (a gene therapy delivery system) using analytical ultracentrifugation. Anal Biochem. 2007;362:16–37.

    Article  CAS  Google Scholar 

  39. Mass photometry of AAVs. Refeyn Application Note 2020.

  40. Lock M, McGorray S, Auricchio A, Ayuso E, Beecham EJ, Blouin-Tavel V, et al. Characterization of a recombinant adeno-associated virus type 2 reference standard material. Hum Gene Ther. 2010;21:1273–85.

    Article  CAS  Google Scholar 

  41. Fu X, Chen WC, Argento C, Clarner P, Bhatt V, Dickerson R, et al. Analytical strategies for quantification of adeno-associated virus empty capsids to support process development. Hum Gene Ther Methods. 2019;30:144–52.

    Article  CAS  Google Scholar 

  42. Wang C, Mulagapati SHR, Chen Z, Du J, Zhao X, Xi G, et al. Developing an anion exchange chromatography assay for determining empty and full capsid contents in AAV6.2. Mol Ther Methods Clin Dev. 2019;15:257–63.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by the intramural program of the NHLBI, NIH. We thank Suja Hiriyanna (Ocular Gene Therapy Core, NEI, NIH) for generating the virus constructs.

Funding

This work was supported by the Intramural Research Program of the NHLBI, NIH (HL000338).

Author information

Authors and Affiliations

  1. Biophysics Core Facility, National Heart, Lung, and Blood Institute, 50 South Drive, Bethesda, MD, 20892-8012, USA

    Di Wu & Grzegorz Piszczek

  2. Ocular Gene Therapy Core Facility, National Eye Institute, 6 Center Drive, Bethesda, MD, 20892, USA

    Philsang Hwang & Tiansen Li

Authors
  1. Di Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Philsang Hwang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tiansen Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Grzegorz Piszczek
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

DW and GP conceived and planned the experiments and designed and generated the figures; DW performed the experiments and analyzed the data; PH and TL designed and produced the rAAV vectors; and GP wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Grzegorz Piszczek.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Wu, D., Hwang, P., Li, T. et al. Rapid characterization of adeno-associated virus (AAV) gene therapy vectors by mass photometry. Gene Ther 29, 691–697 (2022). https://doi.org/10.1038/s41434-021-00311-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41434-021-00311-4

Search

Advanced search

Quick links