Assessment of the percentage of full recombinant adeno-associated virus particles in a gene therapy drug using CryoTEM.

In spite of continuous development of gene therapy vectors with thousands of drug candidates in clinical drug trials there are only a small number approved on the market today stressing the need to have characterization methods to assist in the validation of the drug development process. The level of packaging of the vector capsids appears to play a critical role in immunogenicity, hence an objective quantitative method assessing the content of particles containing a genome is an essential quality measurement. As transmission electron microscopy (TEM) allows direct visualization of the particles present in a specimen, it naturally seems as the most intuitive method of choice for characterizing recombinant adeno-associated virus (rAAV) particle packaging. Negative stain TEM (nsTEM) is an established characterization method for analysing the packaging of viral vectors. It has however shown limitations in terms of reliability. To overcome this drawback, we propose an analytical method based on CryoTEM that unambiguously and robustly determines the percentage of filled particles in an rAAV sample. In addition, we show that at a fixed number of vector particles the portion of filled particles correlates well with the potency of the drug. The method has been validated according to the ICH Q2 (R1) guidelines and the components investigated during the validation are presented in this study. The reliability of nsTEM as a method for the assessment of filled particles is also investigated along with a discussion about the origin of the observed variability of this method.

Introduction of the hybcell-based compact sequencing technology and comparison to state-of-the-art methodologies for KRAS mutation detection.

The detection of KRAS mutations in codons 12 and 13 is critical for anti-EGFR therapy strategies; however, only those methodologies with high sensitivity, specificity, and accuracy as well as the best cost and turnaround balance are suitable for routine daily testing. Here we compared the performance of compact sequencing using the novel hybcell technology with 454 next-generation sequencing (454-NGS), Sanger sequencing, and pyrosequencing, using an evaluation panel of 35 specimens. A total of 32 mutations and 10 wild-type cases were reported using 454-NGS as the reference method. Specificity ranged from 100% for Sanger sequencing to 80% for pyrosequencing. Sanger sequencing and hybcell-based compact sequencing achieved a sensitivity of 96%, whereas pyrosequencing had a sensitivity of 88%. Accuracy was 97% for Sanger sequencing, 85% for pyrosequencing, and 94% for hybcell-based compact sequencing. Quantitative results were obtained for 454-NGS and hybcell-based compact sequencing data, resulting in a significant correlation (r = 0.914). Whereas pyrosequencing and Sanger sequencing were not able to detect multiple mutated cell clones within one tumor specimen, 454-NGS and the hybcell-based compact sequencing detected multiple mutations in two specimens. Our comparison shows that the hybcell-based compact sequencing is a valuable alternative to state-of-the-art methodologies used for detection of clinically relevant point mutations.

Insulin hypersensitivity induced by hepatic PTEN gene ablation protects from murine endotoxemia.

Sepsis still remains a major cause for morbidity and mortality in patients. The molecular mechanisms underlying the disease are still enigmatic. A great number of therapeutic approaches have failed and treatment strategies are limited to date. Among those few admitted for clinical intervention, intensive insulin treatment has proven to be effective in the reduction of disease related complications in critically ill patients. Insulin effectively reduces glucose levels and thereby contributes to protection. On the other hand insulin is a potent signaling pathway activator. One of those is the PI3K signaling axis. Activation of PI3K is known to limit pro-inflammatory gene expression. Here we can show that in a mouse model of insulin hypersensitivity induced by the deletion of the PI3K antagonist PTEN, specifically in hepatic tissue, significant protection is conferred in murine models of lethal endotoxemia and sepsis. Acute inflammatory responses are diminished, glucose metabolism normalized and vascular activation is reduced. Furthermore we investigated the hepatic gene expression profile of relevant anti-inflammatory genes in PTEN deficient mice and found marked upregulation of PPARγ and HO-1. We conclude from our data that insulin hypersensitivity via sustained activation of the PI3K signaling pathway exerts protective effects in acute inflammatory processes.