AAV capsid stability

The problem

Keeping AAV capsids stable is critical to getting genetic payloads where they need to go – since capsids can break down or aggregate during manufacturing or storage. Even after administration, capsid stability is an important part of making sure your AAV releases its payload exactly when it’s supposed to.

Measuring when an AAV breaks open or aggregates is a problem that usually requires answers from many tools put together, and techniques can be complex, take a long time to run, or require large sample volumes. Functional assays, like transduction or infectivity assays, take days and require highly skilled users, while electron microscopy is low throughput and expensive. Even routine assays like AAV ELISAs, which can be used to measure intact capsid titers, or qPCR, which looks at genome quantification, can take several hours and will only give insight on capsid stability as part of an intricately designed experiment.

The right tool for the job

Uncle is a one-of-a-kind solution to look at capsid stability in a simple, fast, and low volume assay. Uncle combines thermal ramp studies with full-spectrum fluorescence and static light scattering (SLS) to give stability insights that look at capsid, protein and DNA behavior - all on the same instrument and in the same experiment. Results take less than 2 hours on just 9 μL so you can screen more candidates, formulations, or conditions and quickly figure out which is the most stable.

The proof

See it for the first time

Combine AAV and SYBR Gold® dye in a differential scanning fluorimetry (DSF) experiment on Uncle, and you can pinpoint the temperatures when your AAV capsid starts to lose its genome. This is called genome ejection (or AAV uncoating) and happens with AAV well before capsid proteins unfold and aggregate. SYBR Gold fluorescence intensity increases when there’s more free DNA – either from degraded AAV at the start of an experiment, or as DNA is ejected from AAV capsids during a thermal ramp. The temperature when aggregation starts can be easily tracked by looking for a sharp increase in SLS intensity.

See it for the first time
    Capsid stability depends on a lot:

  • serotype or recombinant capsid sequence
  • packaged genome length
  • if genomes are single-stranded or self-complementary
  • formulation buffer
  • capsid chemical stability, e.g. deamidation

Uncle helps efficiently assess the impact of each of those on stability for an AAV gene therapy vector, and gives insight into the many ways they can impact stability by detecting genome ejection, capsid protein unfolding and aggregation.

Comparing serotypes

Serotypes and recombinant capsids all have different genome ejection behavior. Uncle shows exactly when temperature starts causing problems to help you pick out the best-behaved AAV.

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Optimizing formulations

The best formulation buffers will be designed to help AAV capsids hold in their genomes as long as possible. From excipients to pH, formulations have a lot going on that can help or hinder capsid stability.

Optimizing Formulations_R4
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Cracking stability using a pile of one-trick, protein-hungry tools is a ton of work. Uncle combines 3 different measurement modes — fluorescence, Static Light Scattering (SLS) and Dynamic Light Scattering (DLS). So you can crank out all your data in just a few hours, and use way less protein. All the info you’ll get makes picking the best formulation, protein, or viral vector a piece of cake.

Want more info?

Want to learn more about how Uncle gives unique insights on viral capsid stability?