Analysis of the impact of pluronic acid on the thermal stability and infectivity of AAV6.2FF.

BACKGROUND: The advancement of AAV vectors into clinical testing has accelerated rapidly over the past two decades. While many of the AAV vectors being utilized in clinical trials are derived from natural serotypes, engineered serotypes are progressing toward clinical translation due to their enhanced tissue tropism and immune evasive properties. However, novel AAV vectors require formulation and stability testing to determine optimal storage conditions prior to their use in a clinical setting. RESULTS: Here, we evaluated the thermal stability of AAV6.2FF, a rationally engineered capsid with strong tropism for lung and muscle, in two different buffer formulations; phosphate buffered saline (PBS), or PBS supplemented with 0.001% non-ionic surfactant Pluronic F68 (PF-68). Aliquots of AAV6.2FF vector encoding the firefly luciferase reporter gene (AAV6.2FF-ffLuc) were incubated at temperatures ranging from -20°C to 55°C for varying periods of time and the impact on infectivity and particle integrity evaluated. Additionally, the impact of several rounds of freeze-thaw treatments on the infectivity of AAV6.2FF was investigated. Vector infectivity was measured by quantifying firefly luciferase expression in HEK 293 cells and AAV particle integrity was measured by qPCR quantification of encapsidated viral DNA. CONCLUSIONS: Our data demonstrate that formulating AAV6.2FF in PBS containing 0.001% PF-68 leads to increased stability and particle integrity at temperatures between -20℃ to 21℃ and protection against the destructive effects of freeze-thaw. Finally, AAV6.2FF-GFP formulated in PBS supplemented with 0.001% PF-68 displayed higher transduction efficiency in vivo in murine lung epithelial cells following intranasal administration than vector buffered in PBS alone further demonstrating the beneficial properties of PF-68.

Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models.

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. Clinical advancement of novel applications for AAV involves transitioning from small animal models, such as mice, to larger animal models, including dogs, sheep, and nonhuman primates. One of the limitations of administering AAV to larger animals is the requirement for large quantities of high-titer virus. While suspension cell culture is a scalable method for AAV vector production, few research labs have the equipment (e.g., bioreactors) or know how to produce AAV in this manner. Moreover, AAV titers are often significantly lower when produced in suspension HEK 293 cells as compared to adherent HEK293 cells. Described here is a method for producing large quantities of high-titer AAV using cell stacks. A detailed protocol for titering AAV as well as methods for validating vector purity are also described. Finally, representative results of AAV-mediated transgene expression in a sheep model are presented. This optimized protocol for large-scale production of AAV vectors in adherent cells will enable molecular biology laboratories to advance the testing of their novel AAV therapies in larger animal models.

Safety and Tolerability of the Adeno-Associated Virus Vector, AAV6.2FF, Expressing a Monoclonal Antibody in Murine and Ovine Animal Models.

Adeno-associated virus (AAV) vector mediated expression of therapeutic monoclonal antibodies is an alternative strategy to traditional vaccination to generate immunity in immunosuppressed or immunosenescent individuals. In this study, we vectorized a human monoclonal antibody (31C2) directed against the spike protein of SARS-CoV-2 and determined the safety profile of this AAV vector in mice and sheep as a large animal model. In both studies, plasma biochemical parameters and hematology were comparable to untreated controls. Except for mild myositis at the site of injection, none of the major organs revealed any signs of toxicity. AAV-mediated human IgG expression increased steadily throughout the 28-day study in sheep, resulting in peak concentrations of 21.4-46.7 µg/ mL, demonstrating practical scale up from rodent to large animal models. This alternative approach to immunity is worth further exploration after this demonstration of safety, tolerability, and scalability in a large animal model.