DNA leaks before capsids pop: AAV thermal stability on Uncle

Importance of the Topic

Understanding the thermal stability of adeno-associated virus (AAV) vectors is critical for developing robust gene therapies. Thermal stress can trigger genome ejection or capsid disruption, affecting vector potency, storage, and formulation. Rapid, multi-parameter assays that reveal capsid behavior, genome integrity, and aggregation can greatly accelerate formulation screening and quality control in both research and regulated environments.

Study Goals and Overview

This application note demonstrates how the Uncle multi-modal stability platform characterizes AAV thermal stability by simultaneously monitoring three degradation pathways: genome ejection, capsid unfolding, and particle aggregation. Experiments profile different serotypes, concentrations, and buffer conditions, showcasing how to extract melting temperatures (Tm), aggregation onset (Tagg), and particle counts in a single two-hour workflow.

Methodology and Instrumentation

• Platform: Uncle stability system with full-spectrum fluorescence, static light scattering (SLS), and dynamic light scattering (DLS).
• Temperature range: 15–95 °C; heating rate 0.5 °C/min in sealed quartz cuvettes (9 µL sample, up to 48 wells).
• Fluorescence probes: SYBR Gold (473 nm excitation) for DNA release; intrinsic protein fluorescence (266 nm excitation) for capsid unfolding.
• Aggregation metrics: SLS Tagg at 266 nm and 473 nm; DLS size distributions for pre- and post-ramp aggregation assessment and particle count estimation.
• Samples: AAV1, AAV2, AAV9 vectors expressing CMV-GFP (5 × 10¹¹–1 × 10¹⁴ vg/mL) in PBS-based formulations with optional excipients (Pluronic F68, sodium citrate, arginine).

Main Results and Discussion

• Genome ejection Tm for AAV9 consistently around 57 °C across concentrations (5 × 10¹¹ to 1 × 10¹³ vg/mL), indicating minimal concentration dependence.
• Capsid unfolding Tm by intrinsic fluorescence measured for AAV9 at ~78.6 °C, in close agreement with literature values obtained by SYPRO Orange DSF. Aggregation onset (Tagg) occurred ~74–75 °C, coinciding with unfolding.
• Serotype comparison: AAV1 (Tm ~83 °C), AAV2 (Tm ~67 °C), AAV9 (Tm ~78 °C) for capsid unfolding; genome ejection stability similarly ranked (AAV9 > AAV1 > AAV2).
• Excipient effects: Addition of 300 mM arginine lowered genome ejection Tm by ~12 °C and reduced capsid unfolding stability, highlighting formulation impacts on viral stability.
• Quantification of free and released DNA via SYBR Gold before and after thermal ramp correlated linearly with genome copy titer, enabling rapid assessment of contaminant or encapsulated DNA.
• Particle counts derived from DLS intensity correlated strongly (R² > 0.995) with manufacturer-supplied titers down to ~5 × 10¹¹ vg/mL, offering a fast alternative to traditional titration methods.

Practical Benefits and Applications

• Consolidates multiple assays (genome release, capsid unfolding, aggregation, particle counting) into one platform and experiment.
• Rapid turnaround (<2 hours) accelerates formulation screening, serotype selection, and stability profiling.
• Small sample volumes (9 µL) and multi-well throughput support efficient use of precious viral material.
• 21 CFR Part 11–compliant software ensures secure data tracking for regulated labs.

Future Trends and Applications

• Integration of further orthogonal measurements (e.g., higher-resolution light scattering) for deeper mechanistic insight into capsid dynamics.
• Automated screening of buffer libraries and excipient combinations to optimize long-term storage stability.
• Extension to other viral vectors and virus-like particles for broader gene therapy and vaccine development workflows.
• Combination with high-throughput functional assays to correlate biophysical metrics with infectivity and transduction efficiency.

Conclusion

The Uncle platform delivers comprehensive, high-throughput thermal stability profiles for AAV vectors, distinguishing genome ejection, capsid unfolding, and aggregation in a single experiment. Its sensitivity, speed, and compliance features make it an invaluable tool for gene therapy development, enabling rapid formulation optimization, serotype comparison, and quality control.

Reference

• Bernaud J. et al., J Biol Phys, 2018, 44(2):181–94.
• Horowitz E. et al., J Virol, 2013, 87(6):2994–3002.
• Bennett A. et al., Mol Ther Methods Clin Dev, 2017, 6:171–82.
• Wright J.F., Gene Ther, 2008.
• Wright J.F. et al., Mol Ther, 2005, 12(1):171–8.