Characterization and quantification of adeno-associated virus capsid-loading states by multi-wavelength analytical ultracentrifugation with UltraScan

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References

• 1. Ma CC, Wang ZL, Xu T, He ZY, Wei YQ. The approved gene therapy drugs worldwide: from 1998 to 2019. Biotechnol. Adv. 40, 107502 (2020).
  Medline CASGoogle Scholar
• 2. US Food and Drug Administration. Approved cellular and gene therapy products. www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellular-and-gene-therapy-products
  Google Scholar
• 3. ClinicalTrials.gov. Gene therapy clinical trials. https://clinicaltrials.gov/ct2/results?cond=&term=gene+therapy&cntry=&state=&city=&dist=
  Google Scholar
• 4. Wang D, Tai PW, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat. Rev. Drug Discov. 18, 358–378 (2019).
  Medline CASGoogle Scholar
• 5. Li C, Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nat. Rev. Genet. 21, 255–272 (2020).
  Medline CASGoogle Scholar
• 6. Wörner TP, Bennet A, Habka S et al. Adeno-associated virus capsid assembly is divergent and stochastic. Nat. Commun. 12, 1642 (2021).
  MedlineGoogle Scholar
• 7. Pei X, Earley LF, He Y et al. Efficient capsid antigen presentation from adeno-associated virus empty virions in vivo. Front. Immunol. 9, 844 (2018).
  MedlineGoogle Scholar
• 8. Wright JF. Product-related impurities in clinical-grade recombinant AAV vectors: characterization and risk assessment. Biomedicines 2, 80–97 (2014).
  MedlineGoogle Scholar
• 9. Colomb-Delsuc M, Raim R, Fiedler C et al. Assessment of the percentage of full recombinant adeno-associated virus particles in a gene therapy drug using CryoTEM. PLOS ONE 17(6), e0269139 (2022).
  Medline CASGoogle Scholar
• 10. Wu D, Hwang P, Li T, Piszczek G. Rapid characterization of adeno-associated virus (AAV) gene therapy vectors by mass photometry. Gene Ther. 29(12), 691–697 (2022).
  Medline CASGoogle Scholar
• 11. Gimpel AL, Katsikis G, Sha S et al. Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies. Mol. Ther. Methods Clin. Dev. 20, 740–754 (2021).
  Medline CASGoogle Scholar
• 12. Mayginnes JP, Reed SE, Berg HG et al. 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 (2006).
  Medline CASGoogle Scholar
• 13. Fagone P, Wright JF, Nathwani AC et al. Systemic errors in quantitative polymerase chain reaction titration of self-complementary adeno-associated viral vectors and improved alternative methods. Hum. Gene Ther. Methods 23, 1–7 (2012).
  Medline CASGoogle Scholar
• 14. Yarawsky AE, Zai-Rose V, Cunningham HM et al. AAV analysis by sedimentation velocity analytical ultracentrifugation: beyond empty and full capsids. Eur. Biophys. J. 52(4–5), 353–366 (2023).
  Medline CASGoogle Scholar
• 15. Hayes DB, Dobnik D. Commentary: multiplex dPCR and SV-AUC are promising assays to robustly monitor the critical quality attribute of AAV drug product integrity. J. Pharm. Sci. 111(8), 2143–2148 (2022).
  Medline CASGoogle Scholar
• 16. Savelyev A, Gorbet GE, Henrickson A, Demeler B. Moving analytical ultracentrifugation software to a good manufacturing practices (GMP) environment. PLOS Comput. Biol. 16, e1007942 (2020).
  Medline CASGoogle Scholar
• 17. Werle AK, Powers TW, Zobel JF et al. Comparison of analytical techniques to quantitate the capsid content of adeno-associated viral vectors. Mol. Ther. Methods Clin. Devel. 23, 254–262 (2021).
  CASGoogle Scholar
• 18. Richter K, Wurm C, Strasser K et al. Purity and DNA content of AAV capsids assessed by analytical ultracentrifugation and orthogonal biophysical techniques. Eur. J. Pharm. Biopharm. 189, 68–83 (2023).
  Medline CASGoogle Scholar
• 19. Berkowitz SA, Philo JS. Monitoring the homogeneity of adenovirus preparations (a gene therapy delivery system) using analytical ultracentrifugation. Anal. Biochem. 362, 16–37 (2007).
  Medline CASGoogle Scholar
• 20. Gorbet GE, Pearson JZ, Demeler AK, Cölfen H, Demeler B. Next-generation AUC: analysis of multiwavelength analytical ultracentrifugation data. Methods Enzymol. 562, 27–47 (2015).
  Medline CASGoogle Scholar
• 21. Henrickson A, Gorbet GE, Savelyev A et al. Multi-wavelength analytical ultracentrifugation of biopolymer mixtures and interactions. Anal. Biochem. 652, 114728 (2022). •• The multiwavelength analytical ultracentrifugation (AUC) technology is explained here and provides a fundamental basis for the understanding of the current manuscript.
  Medline CASGoogle Scholar
• 22. Khasa H, Kilby G, Chen X, Wang C. Analytical band centrifugation for the separation and quantification of empty and full AAV particles. Mol. Ther. Methods Clin. Dev. 21, 585–591 (2021).
  Medline CASGoogle Scholar
• 23. Maruno T, Ishii K, Torisu T, Uchiyama S. Size distribution analysis of the adeno-associated virus vector by the c(s) analysis of band sedimentation analytical ultracentrifugation with multiwavelength detection. J. Pharm. Sci. 112, 937–946 (2023).
  Medline CASGoogle Scholar
• 24. Maruno T, Usami K, Ishii K, Torisu T, Uchiyama S. Comprehensive size distribution and composition analysis of adeno-associated virus vector by multiwavelength sedimentation velocity analytical ultracentrifugation. J. Pharm. Sci. 110, 3375–3384 (2021).
  Medline CASGoogle Scholar
• 25. Horne CR, Henrickson A, Demeler B, Dobson RC. Multi-wavelength analytical ultracentrifugation as a tool to characterise protein–DNA interactions in solution. Eur. Biophys. J. 49, 819–827 (2020).
  Medline CASGoogle Scholar
• 26. Horne CR, Venugopal H, Panjikar S et al. Mechanism of NanR gene repression and allosteric induction of bacterial sialic acid metabolism. Nat. Commun. 12, 1988 (2021).
  Medline CASGoogle Scholar
• 27. Gabir H, Gupta M, Meier M et al. Investigation of dynamic solution interactions between NET-1 and UNC-5B by multi-wavelength analytical ultracentrifugation. Eur. Biophys. J. 52(4–5), 473–481 (2023).
  Medline CASGoogle Scholar
• 28. Ahmed I, Hahn J, Henrickson A et al. Structure–function studies reveal ComEA contains an oligomerization domain essential for transformation in Gram-positive bacteria. Nat. Commun. 13, 7724 (2022).
  Medline CASGoogle Scholar
• 29. Demeler B, Gorbet GE. Chapter 8: analytical ultracentrifugation data analysis with UltraScan-III. In: Analytical Ultracentrifugation: Instrumentation, Software, and Applications. Uchiyama SArisaka FStafford WLaue T (Eds). Springer, Tokyo, 119–143 (2016). • The UltraScan software package is a comprehensive hydrodynamic characterization toolkit that is open source and freely available, and used for all analysis performed in this manuscript.
  Google Scholar
• 30. Mortezazadeh S, Demeler B. Systematic noise removal from analytical ultracentrifugation data with UltraScan. Eur. Biophys. J. 52(4–5), 203–213 (2023). •• The pseudo-absorbance conversion module allows intensity to be used for equilibrium experiments and essentially doubles the capacity of the AUC analytical buoyant density equilibrium technique by removing time-invariant and baseline noise contributions.
  MedlineGoogle Scholar
• 31. Savelyev A, Brookes EH, Henrickson A, Demeler B. A new UltraScan module for the characterization and quantification of analytical buoyant density equilibrium experiments to determine AAV capsid loading. Eur. Biophys. J. 52(4–5), 311–320 (2023). •• The analytical buoyant density equilibrium peak integration tool described here is a logical module to be used for the rigorous quantification of adeno-associated virus capid-loading states.
  MedlineGoogle Scholar
• 32. Mortezazadeh S, Demeler B. A spectral decomposition quality assessment tool for multi-wavelength AUC experiments with UltraScan. Eur. Biophys. J. 52(4–5), 303–310 (2023). • The spectral decomposition viewer is used to monitor the multi-wavelength decomposition of AUC data to ensure an adequate fit quality.
  Medline CASGoogle Scholar
• 33. Sternisha SM, Wilson AD, Bouda E, Bhattacharya A, VerHeul R. Optimizing high-throughput viral vector characterization with density gradient equilibrium analytical ultracentrifugation. Eur. Biophys. J. 52(4–5), 387–392 (2023).
  Medline CASGoogle Scholar
• 34. Bepperling A, Best J. Comparison of three AUC techniques for the determination of the loading status and capsid titer of AAVs. Eur. Biophys. J. 52(4–5), 401–413 (2023). Epub 2023 May 28. • The authors reach similar conclusions for various AUC techniques applied to adeno-associated virus characterization.
  Medline CASGoogle Scholar
• 35. Deng S, Oka K. Adeno-associated virus as gene delivery vehicle into the retina. Methods Mol. Biol. 2092, 77–90 (2020).
  Medline CASGoogle Scholar
• 36. Ayuso E, Mingozzi F, Montane J et al. High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency. Gene Ther. 17, 503–510 (2010).
  Medline CASGoogle Scholar
• 37. Challis RC, Kumar SR, Chan KY et al. Systemic AAV vectors for widespread and targeted gene delivery in rodents. Nat. Protoc. 14, 379–414 (2019).
  Medline CASGoogle Scholar
• 38. Kumar SR, Miles TF, Chen X et al. Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types. Nat. Methods 17(5), 541–550 (2020).
  MedlineGoogle Scholar
• 39. Berkowitz SA, Laue T. Boundary convection during sedimentation velocity in the Optima analytical ultracentrifuge. Anal. Biochem. 631, 114306 (2021).
  Medline CASGoogle Scholar
• 40. Demeler B. Methods for the design and analysis of sedimentation velocity and sedimentation equilibrium experiments with proteins. Curr. Protoc. Protein Sci. 60, 7.13.1–7.13.24 (2010).
  Google Scholar
• 41. Brookes EH, Boppana RV, Demeler B. Computing large sparse multivariate optimization problems with an application in biophysics. In: SC'06: Proceedings of the 2006 ACM/IEEE Conference on Supercomputing. IEEE, Tampa, FL, USA, 42–42 (2006).
  Google Scholar
• 42. Brookes E, Cao W, Demeler B. A two-dimensional spectrum analysis for sedimentation velocity experiments of mixtures with heterogeneity in molecular weight and shape. Eur. Biophys. J. 39, 404–414 (2010).
  Google Scholar
• 43. Demeler B, van Holde KE. Sedimentation velocity analysis of highly heterogeneous systems. Anal. Biochem. 335, 279–288 (2004).
  Medline CASGoogle Scholar
• 44. Schuck P, Demeler B. Direct sedimentation analysis of interference optical data in analytical ultracentrifugation. Biophys. J. 76, 2288–2296 (1999).
  Medline CASGoogle Scholar
• 45. Cao W, Demeler B. Modeling analytical ultracentrifugation experiments with an adaptive space–time finite element solution of the Lamm equation. Biophys. J. 89, 1589–1602 (2005).
  Medline CASGoogle Scholar
• 46. Cao W, Demeler B. Modeling analytical ultracentrifugation experiments with an adaptive space–time finite element solution for multicomponent reacting systems. Biophys. J. 95, 54–65 (2008).
  Medline CASGoogle Scholar
• 47. Burnham B, Nass S, Kong E et al. Analytical ultracentrifugation as an approach to characterize recombinant adeno-associated viral vectors. Hum. Gene Ther. Methods 26, 228–242 (2015).
  Medline CASGoogle Scholar
• 48. Fu X, Chen WC, Argento C et al. Analytical strategies for quantification of adeno-associated virus empty capsids to support process development. Hum. Gene Ther. Methods 30, 144–152 (2019).
  Medline CASGoogle Scholar
• 49. Bernaud J, Rossi A, Fis A et al. Characterization of AAV vector particle stability at the single-capsid level. J. Biol. Phys. 44, 181–194 (2018).
  Medline CASGoogle Scholar
• 50. Ros C, Baltzer C, Mani B, Kempf C. Parvovirus uncoating in vitro reveals a mechanism of DNA release without capsid disassembly and striking differences in encapsidated DNA stability. Virology 345, 137–147 (2006).
  Medline CASGoogle Scholar