Fig. 6: In vivo performance of AAV stabilized in a thin film for 30 days at 4 °C.

Mice were given 1.5 × 10¹¹ virus genomes of formulated AAV9 CBA-Luc by tail vein injection. a Thirty days after administration, animals were sacrificed, organs harvested, and assessed for luciferase transgene expression with bioluminescence imaging. Mean (±standard error of the mean) values for total flux for each organ collected from five mice per treatment group are shown. b Bioluminescent images of mice from each treatment group at 1, 8, 15, 22, and 29 days. Relative bioluminescence intensity is shown in pseudo-color, with red and blue representing the strongest and weakest photon fluxes, respectively. NOTE: Two animals, one from the saline control group (Group 1) and that which was given freshly purified vector in the Standard Control Formulation (Group 2), were found deceased in their cages on day 22 and day 29, respectively, with no gross abnormalities observed externally nor internally upon visual inspection of the organs. These deaths were attributed to the blood collection technique that was used at the time. The procedure was immediately changed when these observations were noted so that all mice in the remaining treatment groups were able to complete the full course of the study. Treatment Groups: Group 1: Vehicle (saline control); Group 2: FFF, Group 3: RT, FFF, Group 4: F1S, Group 5: F2S. c Organs with high transgene expression were further analyzed for virus genome copies by quantitative real-time PCR. Data reflect the mean (±standard error of the mean) values obtained from tissues collected from five mice per treatment group. Significant differences between animals treated with vectors in the standard control (FFF) and the F1S and F2S formulations were evaluated by one-way ANOVA with Dunnett’s multiple comparison tests. *p < 0.05. Abbreviations (Panels a and c): Vehicle: saline control, FFF frozen standard control formulation, RT, FFF freshly purified vector in standard control formulation held at room temperature (RT) during the time needed for film formation to be complete then refrozen, F1S high-viscosity film formulation, F2S low-viscosity film formulation, Diaph diaphragm, Spinal Spinal cord, Gastroc gastrocnemius muscle.