Methodological Validation of Sedimentation Velocity Analytical Ultracentrifugation Method for Adeno-Associated Virus and Collaborative Calibration of System Suitability Substance.

Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term in vivo gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio. This includes specificity, accuracy, precision, linearity, and parameter variables involving the sedimentation velocity analytical ultracentrifugation (SV-AUC) method. Specifically, SV-AUC differentiated among the empty, partial, full, and high sedimentation coefficient substance (HSCS) AAV particles while evaluating their sedimentation heterogeneity. The intermediate precision analysis of HE (high percentage of empty capsid) and HF (high percentage of full capsid) samples revealed that the specific species percentage, such as empty or full, was more significant than 50%. Moreover, the relative standard deviation (RSD) could be within 5%. Even for empty or partially less than 15%, the RSD could be within 10%. The accuracy recovery rates of empty capsid were between 103.9% and 108.7% across three different mixtures. When the measured percentage of specific species was more significant than 14%, the recovery rate was between 77.9% and 106.6%. Linearity analysis revealed an excellent linear correlation between the empty, partial, and full in the HE samples. The AAV samples with as low as 7.4 × 1011 cp/mL AAV could be accurately quantified with SV-AUC. The parameter variable analyses revealed that variations in cell alignment significantly affected the overall results. Still, the detection wavelength of 235 nm slightly influenced the empty, partial, and full percentages. Minor detection wavelength changes showed no impact on the sedimentation coefficient of these species. However, the temperature affected the measured sedimentation coefficient. These results validated the SV-AUC method to quantify AAV. This study provides solutions to AAV empty and full capsid ratio quantification challenges and the subsequent basis for calibrating the AAV empty capsid system suitability substance. Because of the AAV structure and potential variability complexity in detection, we jointly calibrated empty capsid system suitability substance with three laboratories to accurately detect the quantitative AAV empty and full capsid ratio. The empty capsid system suitability substance could be used as an external reference to measure the performance of the instrument. The results could be compared with multiple QC (quality control) laboratories based on the AAV vector and calibration accuracy. This is crucial for AUC to be used for QC release and promote gene therapy research worldwide.

Interaction with Ppil3 leads to the cytoplasmic localization of Apoptin in tumor cells.

Apoptin, a small protein encoded by chicken anemia virus (CAV), induces cell death specifically in cancer cells. In normal cells, Apoptin remains in the cytoplasm; whereas in cancerous cells, it migrates into the nucleus and kills the cell. Cellular localization appears to be crucial. Through a yeast two-hybrid screen, we identified human Peptidyl-prolyl isomerase-like 3 (Ppil3) as one of the Apoptin-associated proteins. Ppil3 could bind Apoptin directly, and held Apoptin in cytoplasm even in tumor cells. We then demonstrated that the nuclearcytoplasmic distribution of Apoptin is related to the expression level of intrinsic Ppil3. Moreover, extrinsic modifying of Ppil3 levels also resulted in nuclearcytoplasmic shuffling of Apoptin. The Apoptin P109A mutant, located between the putative nuclear localization and export signals, could significantly impair the function of Ppil3. Our results suggest a new direction for the localization mechanism study of Apoptin in cells.

Human macrophages promote the motility and invasiveness of osteopontin-knockdown tumor cells.

Increasing evidence indicates that macrophages in tumor stroma can significantly modify the malignant phenotypes of tumors. Osteopontin (OPN) is frequently overexpressed in cancers with high metastatic capacity and, thus, has been considered as a potential therapeutic target. To find out whether macrophages can affect the outcome of OPN-knockdown tumor cells, we used RNA interference (RNAi) to stably silence the OPN expression in the highly invasive human hepatoma cell line SK-Hep-1. Silencing of OPN markedly decreased the motility and invasiveness of the SK-Hep-1 cells. Further studies using this cell model revealed that coculture with human macrophages or macrophage-conditioned medium largely restored the migration and invasion potential of OPN-knockdown tumor cells. Moreover, such macrophage-promoted motility can be effectively blocked either by the addition of OPN-neutralizing antibody to the cocultured medium or by silencing OPN expression in macrophages. These results indicate that macrophage-derived OPN can compensate for the decrease of OPN and thereby restore the metastatic potential of OPN-knockdown tumor cells. Further characterization of the underlying mechanisms disclosed that macrophage-derived OPN exerted its function independently of the actin cytoskeleton rearrangement or the activation of matrix metalloproteinase and Rho families. Our results suggest that there are fine-tuned complex interactions between cancer cells and stroma cells, which may modify the outcome of cancer therapy, and therefore should be considered for the rational design of anticancer strategy.