Adeno-associated virus vectors for human gene therapy

Adeno-associated virus(AAV) is a small,non-enveloped virus that contains a single-stranded DNA genome. It was the first gene therapy drug approved in the Western world in November 2012 to treat patients with lipoprotein lipase deficiency. AAV made history and put human gene therapy in the forefront again. More than four decades of research on AAV vector biology and human gene therapy has generated a huge amount of valuable information. Over 100 AAV serotypes and variants have been isolated and at least partially characterized. A number of them have been used for preclinical studies in a variety of animal models. Several AAV vector production platforms,especially the baculovirus-based system have been established for commercial-scale AAV vector production. AAV purification technologies such as density gradient centrifugation,column chromatography,or a combination,have been well developed. More than 117 clinical trials have been conducted with AAV vectors. Although there are still challenges down the road,such as crossspecies variation in vector tissue tropism and gene transfer efficiency,pre-existing humoral immunity to AAV capsids and vector dose-dependent toxicity in patients,the gene therapy community is forging ahead with cautious optimism. In this review I will focus on the properties and applications of commonly used AAV serotypes and variants,and the technologies for AAV vector production and purification. I will also discuss the advancement of several promising gene therapy clinical trials.

Efficient purification of cell culture-derived classical swine fever virus by ultrafiltration and size-exclusion chromatography

Large-scale production of cell culture-based classical swine fever virus(CSFV)vaccine is hampered by the adverse reactions caused by contaminants from host cell and culture medium.Hence,we have developed an efficient method for purifying CSFV from cell-culture medium.Pure viral particles were obtained with two steps of tangential-flow filtration(TFF)and size-exclusion chromatography(SEC),and were compared with particles from ultracentrifugation by transmission electron microscopy(TEM),infectivity and recovery test,and real time fluorescent quantitative PCR(FQ-PCR).TFF concentrated the virus particles effectively with a retention rate of 98.5%,and 86.2%of viral particles were obtained from the ultrafiltration retentate through a Sepharose 4 F F column on a biological liquid chromatography system.CSFV purified by TFF-SEC or ultracentrifugation were both biologically active from 1.0×10–4.25 TCID50·mL^(–1) to 3.0×10^(–6.25) TCID50·mL^(–1),but the combination of TFF and SEC produced more pure virus particles than by ultracentrifugation alone.In addition,pure CSFV particles with the expected diameter of 40–60 nm were roughly spherical without any visible contamination.Mice immunized with CSFV purified by TFF-SEC produced higher antibody levels compared with immunization with ultracentrifugation-purified CSFV(P<0.05).The purification procedures in this study are reliable technically and feasible for purification of large volumes of viruses.

A nanofilter composed of carbon nanotube-silver composites for virus removal and antibacterial activity improvement

We have developed a new nanofilter using a carbon nanotube-silver composite material that is capable of efficiently removing waterborne viruses and bacteria.The nanofilter was subjected to plasma surface treatment to enhance its flow rate,which was improved by approximately 62%.Nanoscale pores were obtained by fabricating a carbon nanotube network and using nanoparticle fixation technology for the removal of viruses.The pore size of the nanofilter was approximately 38 nm and the measured flow rate ranged from 21.0 to 97.2 L/（min·m^2）under a pressure of 1–6 kgf/cm^2 when the amount of loaded carbon nanotube-silver composite was 1.0 mg/cm^2.The nanofilter was tested against Polio-,Noro-,and Coxsackie viruses using a sensitive real-time polymerase chain reaction assay to detect the presence of viral particles within the outflow.No trace of viruses was found to flow through the nanofilter with carbon nanotube-silver composite loaded above 0.8 mg/cm^2.Moreover,the surface of the filter has antibacterial properties to prevent bacterial clogging due to the presence of 20-nm silver nanoparticles,which were synthesized on the carbon nanotube surface.