Thermostability and in vivo performance of AAV9 in a film matrix

Abstract

Background Adeno-associated virus (AAV) vectors are stored and shipped frozen which poses logistic and economic barriers for global access to these therapeutics. To address this issue, we developed a method to stabilize AAV serotype 9 (AAV9) in a film matrix that can be stored at ambient temperature and administered by systemic injection. Methods AAV9 expressing the luciferase transgene was mixed with formulations, poured into molds and films dried under aseptic conditions. Films were packaged in individual particle-free bags with foil overlays and stored at various temperatures under controlled humidity. Recovery of AAV9 from films was determined by serial dilution of rehydrated film in media and infection of HeLa RC32 cells. Luciferase expression was compared to that of films rehydrated immediately after drying. Biodistribution of vector was determined by in vivo imaging and quantitative real-time PCR. Residual moisture in films was determined by Karl Fischer titration. Results AAV9 embedded within a film matrix and stored at 4 °C for 5 months retained 100% of initial titer. High and low viscosity formulations maintained 90 and 85% of initial titer after 6 months at 25 °C respectively. AAV was not detected after 4 months in a Standard Control Formulation under the same conditions. Biodistribution and transgene expression of AAV stored in film at 25 or 4 °C were as robust as vector stored at −80 °C in a Standard Control Formulation. Conclusions These results suggest that storage of AAV in a film matrix facilitates easy transport of vector to remote sites without compromising in vivo performance.

Clinical and Preclinical Methods of Heat-Stabilization of Human Vaccines

“…Another innovative approach is the expression of vaccine elements in yeast, followed by whole-cell lyophilization, to enhance the protection of biological components during the process . Furthermore, lyophilization can be used to create foams through sacrificial excipients that sublimate with the water , and can also form thin films, typically with highly viscous solutions, that can be hydrated prior to injection. , These diverse methods underscore the complexity and adaptability of lyophilization in vaccine production, yet also highlight the need for careful consideration of each vaccine’s unique requirements.…”

“…40 The mixture's viscosity is also changed, which can help protect during the lyophilization process. 30 The increased viscosity better supports formation of a glassy matrix to protect the biologics during the freezing process. Once dried, this glassy matrix forms a protective barrier that provides heat stability.…”

“…By adding cryoprotects, the glass transition temperature of the formulation is increased, which can offer better protein stability as the biologics are frozen and dried . The mixture’s viscosity is also changed, which can help protect during the lyophilization process . The increased viscosity better supports formation of a glassy matrix to protect the biologics during the freezing process.…”

“…Table presents several infectious disease vaccines evaluated preclinically for heat-stabilization. Several viral vectors (e.g., adenovirus, adeno-associated virus) that can be used for infectious disease vaccines, gene therapy, and other applications have also been evaluated for heat stability. ,− …”

Identification of film-based formulations that move mRNA lipid nanoparticles out of the freezer

“…This process has been shown to allow bacteria, enzymes, live adenovirus, adeno-associated virus (AAV), and plasmid DNA to be stored and shipped without refrigeration. 22 , 23 , 24 A secondary objective was to understand how data collected from commonly accepted stability-indicating assays for LNP products can be used to predict bioactivity. Successful identification of a film-based formulation that eliminates the need for ultra-low cold-chain storage and transport and identifying predictors of LNP performance will significantly improve access to these lifesaving medications and readiness for the next pandemic.…”