Characterization of AAV vector particle stability at the single-capsid level


Virus families have evolved different strategies for genome uncoating, which are also followed by recombinant vectors. Vectors derived from adeno-associated viruses (AAV) are considered as leading delivery tools for in vivo gene transfer, and in particular gene therapy. Using a combination of atomic force microscopy (AFM), biochemical experiments, and physical modeling, we investigated here the physical properties and stability of AAV vector particles. We first compared the morphological properties of AAV vectors derived from two different serotypes (AAV8 and AAV9). Furthermore, we triggered ssDNA uncoating by incubating vector particles to increasing controlled temperatures. Our analyses, performed at the single-particle level, indicate that genome release can occur in vitro via two alternative pathways: either the capsid remains intact and ejects linearly the ssDNA molecule, or the capsid is ruptured, leaving ssDNA in a compact entangled conformation. The analysis of the length distributions of ejected genomes further revealed a two-step ejection behavior. We propose a kinetic model aimed at quantitatively describing the evolution of capsids and genomes along the different pathways, as a function of time and temperature. This model allows quantifying the relative stability of AAV8 and AAV9 particles.

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We would like to thank Federico Mingozzi for helpful discussions. This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS; PEPS MPI). It was also funded by grants from the Ecole Normale Supérieure (ENS) de Lyon (to CFM and AS) and Association Française contre les Myopathies (AFM) to AS, HB, and CFM.

Authors’ contributions statement

JB, AR, AS, and CFM conceived and designed the experiments. JB, AF, AR, LG, and AL performed the experiments. JB, AF, AR, MC, HB, AS, and CFM analyzed the data. MC, AS, CFM wrote the paper. .


The authors declare no competing financial interests.

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Correspondence to Martin Castelnovo or Anna Salvetti or Cendrine Faivre-Moskalenko.

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The authors declare that they have no conflict of interest.

Electronic supplementary material


Supplemental Figure 1 Protocol for the analysis of the AAV response to changes in temperature. Heating of AAV capsids is done for a fixed time at various temperatures ranging from RT to 80 °C before quenching on ice. Mg2+ ions are necessary for DNA binding onto mica, they are added after the heating process as divalent ions are expected to have an effect on capsid stability. The solution is incubated for 5 min on the mica to favor electrostatic adsorption of both DNA and AAV virions. Supplemental Fig. 2: AFM topographic image of AAV8 particles and automated image analysis. (a) raw AFM topographic image of AAV8 capsids, (b) binary image resulting from height and area thresholding, and (c) final topographic image after a few more steps including fractal parameter selection. The further measure of diameter, height or asymmetry on several hundreds of capsids analyzed one by one provides a quantitative morphological characterization of AAV. Supplemental Fig. 3: Automated image analysis of the DNA molecules ejected from the destabilized virions. Using a home-made Matlab script, we can remove the capsids from the image (height thresholding) and skeletonize the DNA fragments in order to measure their length distribution as a function of temperature. Each image with its number corresponds to a different step in the analysis procedure. (a) Initial AFM topographic image. (b) The capsids are removed by applying a height threshold that keep only pixels below 2 nm. (c) Image obtained after the virion has been removed and following by erosion of 1 to 2 pixels around the holes created by capsid removal (height color has been changed). (d) Binary image of DNA only obtained by applying a noise threshold (0.2 nm). (e) The skeleton of DNA filaments can be extracted from the binary image by using morphological tools (erosion). (f) Final topographic image corresponds to image in (c) where the DNA skeleton path has been overlapped in red, as it can clearly been distinguished in the image zoom. Supplemental Fig. 4: AFM imaging of AAV9 virion destabilization induced by heating. Typical AFM images for different heating conditions (a) T = 50 °C, (b) T = 60 °C, (c) T = 65 °C, (d) T = 70 °C, (e) T = 75 °C and (f) T = 80 °C. Each image is shown twice: on the right a color scale of 25 nm is used to see the viral capsids, on the left a 5 nm color scale is chosen to explore what is happening near the surface (ss-DNA width is below 1 nm). At T = 50 °C (a) opened capsids are already observed as DNA is visible on the surface. By increasing the temperature (b), (c) and (d), some filaments can still be observed around the capsids. At higher temperatures (e), both free DNA and DNA linked to capsids exist. At 80 °C mostly free DNA is detected on the surface (f). (PDF 5983 kb)

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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).

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  • Capsid disassembly
  • Atomic force microscopy
  • Stochastic forces
  • Genome uncoating