In vivo gene transduction with adeno-associated virus (AAV)-based vectors depends on laborious procedures for the production of high-titer vector stocks. Purification steps for efficient clearance of impurities such as host cell proteins and empty vector particles are required to meet end-product specifications. Therefore, the development of alternative, realistic methods to facilitate a scalable virus recovery procedure is critical to promote in vivo investigations. However, the conventional purification procedure with resin-based packed-bed chromatography suffers from a number of limitations, including variations in pressure, slow pore diffusion, and large bed volumes. Here we have employed disposable high-performance anion- and cation-exchange membrane adsorbers to effectively purify recombinant viruses. As a result of isoelectric focusing analysis, the isoelectric point of empty particles was found to be significantly higher than that of packaged virions. Therefore, AAV vector purification with the membrane adsorbers was successful and allowed higher levels of gene transfer in vivo without remarkable signs of toxicity or inflammation. Electron microscopy of the AAV vector stocks obtained revealed highly purified virions with as few as 0.8% empty particles. Furthermore, the membrane adsorbers enabled recovery of AAV vectors in the transduced culture supernatant. Also, the ion-exchange enrichment of retroviral vectors bearing the amphotropic envelope was successful. This rapid and scalable viral purification protocol using disposable membrane adsorbers is particularly promising for in vivo experimentation and clinical investigations.

Duchenne muscular dystrophy (DMD) is the most common form of the progressive muscular dystrophies characterized by defects of the dystrophin gene. Although primarily characterized by degeneration of the limb muscles, cardiomyopathy is a major cause of death. Therefore, the development of curative modalities such as gene therapy is imperative. We evaluated the cardiomyopathic features of mdx mice to observe improvements in response to intravenous administration of recombinant adeno-associated virus (AAV) type 9 encoding microdystrophin. The myocardium was extensively transduced with microdystrophin to significantly prevent the development of fibrosis, and expression persisted for the duration of the study. Intraventricular conduction patterns, such as the QRS complex duration and S/R ratio in electrocardiography, were also corrected, indicating that the transduced microdystrophin has a protective effect on the dystrophin-deficient myocardium. Furthermore, BNP and ANP levels were reduced to normal, suggesting the absence of cardiac dysfunction. In aged mice, prevention of ectopic beats as well as echocardiographic amelioration was also demonstrated with improved exercise performance. These findings indicate that AAV-mediated cardiac transduction with microdystrophin might be a promising therapeutic strategy for the treatment of dystrophin-deficient cardiomyopathy.

Sibling visits to the NICU did not result in an increase in the nosocomial viral infection rate.

Background: Outbreaks of enterovirus D68 (EV-D68) respiratory infections in children were reported globally in 2014. In Japan, there was an EV-D68 outbreak in the autumn of 2015 (September-October). The aim of this study was to compare EV-D68-specific polymerase chain reaction (PCR)-positive and EV-D68-specific PCR-negative patients. Methods: Pediatric patients admitted for any respiratory symptoms between September and October 2015 were enrolled. Nasopharyngeal swabs were tested for multiplex respiratory virus PCR and EV-D68-specific reverse transcription-PCR. EV-D68-specific PCR-positive and -negative patients were compared regarding demographic data and clinical information. Results: A nasopharyngeal swab was obtained from 76 of 165 patients admitted with respiratory symptoms during the study period. EV-D68 was detected in 40 samples (52.6%). Median age in the EV-D68-specific PCR-positive and -negative groups was 3.0 years (IQR, 5.5 years) and 3.0 years (IQR, 4.0 years), respectively. The rates of coinfection in the two groups were 32.5% and 47.2%, respectively. There was no significant difference in the history of asthma or recurrent wheezing, length of hospitalization, or pediatric intensive care unit admission rate between the groups. The median days between symptom onset and admission was significantly lower for the EV-D68-positive group (3.0 days vs 5.0 days, P = 0.001). EV-D68 was identified as clade B on phylogenetic analysis. No cases of acute flaccid myelitis were encountered. Conclusions: More than half of the samples from the children admitted with respiratory symptoms were positive for EV-D68-specific PCR during the outbreak. Asthma history was not associated with the risk of developing severe respiratory infection.

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