AAV 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 tie up resources, 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
Aunty is a one-of-a-kind solution to look at AAV stability in a simple, fast, and low volume assay. Aunty 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. Have results on up to 96 samples in under 2 hours from just 8 μL per sample, 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 Aunty, 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.
- 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
Aunty 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. Aunty shows exactly when temperature starts causing problems to help you pick out the best-behaved AAV.
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.
Aunty
Aunty is the fastest and highest throughput protein stability characterization tool on the planet. Period. With unrivaled fluorescence, Static Light Scattering (SLS) and Dynamic Light Scattering (DLS) skills, Aunty blasts through thermal melting and aggregation experiments — reading an entire 96-well plate of samples every minute of a thermal ramp. Sift through piles of candidates, conditions, or formulations, with as many replicates as you want. Aunty cranks out plates of results with unprecedented data resolution, so you can find your winners at breakneck speed.
Want more info?
Want to learn more about how Aunty gives unique insights on viral capsid stability?