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Briefings in functional genomics & proteomics (1)
Clinical and Vaccine Immunology : CVI (1)
Journal of virological methods (1)
Microbial Cell Factories (1)
Negrete, Alejandro (4)
Kotin, Robert M. (3)
Cecchini, Sylvain (1)
Cohen, Jeffrey I. (1)
Graham, Barney S. (1)
Ng, Weng-Ian (1)
Shiloach, Joseph (1)
Virag, Tamas (1)
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Glucose uptake regulation in E. coli by the small RNA SgrS: comparative analysis of E. coli K-12 (JM109 and MG1655) and E. coli B (BL21)
Microbial Cell Factories
The effect of high glucose concentration on the transcription levels of the small RNA SgrS and the messenger RNA ptsG, (encoding the glucose transporter IICBGlc), was studied in both E. coli K-12 (MG1655 and JM109) and E. coli B (BL21). It is known that the transcription level of sgrS increases when E. coli K-12 (MG1655 and JM109) is exposed to the non-metabolized glucose alpha methyl glucoside (αMG) or when the bacteria with a defective glycolysis pathway is grown in presence of glucose. The increased level of sRNA SgrS reduces the level of the ptsG mRNA and consequently lowers the level of the glucose transporter IICBGlc. The suggested trigger for this action is the accumulation of the corresponding phospho-sugars.
In the course of the described work, it was found that E. coli B (BL21) and E. coli K-12 (JM109 and MG1655) responded similarly to αMG: both strains increased SgrS transcription and reduced ptsG transcription. However, the two strains reacted differently to high glucose concentration (40 g/L). E. coli B (BL21) reacted by increasing sgrS transcription and reducing ptsG transcription while E. coli K-12 (JM109 and MG1655) did not respond to the high glucose concentration, and, therefore, transcription of sgrS was not detected and ptsG mRNA level was not affected.
The results suggest that E. coli B (BL21) tolerates high glucose concentration not only by its more efficient central carbon metabolism, but also by controlling the glucose transport into the cells regulated by the sRNA SgrS, which may suggest a way to control glucose consumption and increase its efficient utilization.
Evidence of Prior Exposure to Human Bocavirus as Determined by a Retrospective Serological Study of 404 Serum Samples from Adults in the United States▿
Graham, Barney S.
Cohen, Jeffrey I.
Kotin, Robert M.
Clinical and Vaccine Immunology : CVI
Recently, molecular screening for pathogenic agents has identified a partial genome of a novel parvovirus, called human bocavirus (HBoV). The presence of this newly described parvovirus correlated with upper and lower respiratory tract infections in children. Lower respiratory tract infections are a leading cause of hospital admission in children, and the etiological agent has not been identified in up to 39% of these cases. Using baculovirus expression vectors (BEVs) and an insect cell system, we produced virus-like particles (VLPs) of HBoV. The engineered BEVs express the HBoV capsid proteins stoichiometrically from a single open reading frame. Three capsid proteins assemble into the VLP rather than two proteins predicted from the HBoV genome sequence. The denatured capsid proteins VP1, VP2, and VP3 resolve on silver-stained sodium dodecyl sulfate-polyacrylamide gels as three bands with apparent molecular masses of 72 kDa, 68 kDa, and 62 kDa, respectively. VP2 apparently initiates at a GCT codon (alanine) 273 nucleotides downstream from the VP1 start site and 114 nucleotides upstream from the VP3 initiation site. We characterized the stable capsids using physical, biochemical, and serological techniques. We found that the density of the VLP is 1.32 g/cm3 and is consistent with an icosahedral symmetry with approximately a 25-nm diameter. Rabbit antiserum against the capsid of HBoV, which did not cross-react with adeno-associated virus type 2, was used to develop enzyme-linked immunosorbent assays (ELISAs) for anti-HBoV antibodies in human serum. Using ELISA, we tested 404 human serum samples and established a range of antibody titers in a large U.S. adult population sample.
Strategies for manufacturing recombinant adeno-associated virus vectors for gene therapy applications exploiting baculovirus technology
Kotin, Robert M.
Briefings in functional genomics & proteomics
The development of recombinant adeno-associated virus (rAAV) gene therapy applications is hampered by the inability to produce rAAV in sufficient quantities to support pre-clinical and clinical trials. Contrasting with adherent cell cultures, suspension cultures provide a straightforward means for expansion, however, transiently expressing the necessary, but cytotoxic virus proteins remains the challenge for rAAV production. Both the expansion and expression issues are resolved by using the baculovirus expression vector (BEV) and insect cell culture system. This review addresses strategies for the production of rAAV exploiting baculovirus technology at different scales using different configurations of bioreactors as well as processing and product characterization issues. The yields obtained with these optimized processes exceed ~1 × 1014 vector particles per liter of cell culture suitable for pre-clinical and clinical trials and possible commercialization.
adeno-associated vectors; gene therapy; large-scale production; baculovirus; insect cell
Production of recombinant adeno-associated vectors using two bioreactor configurations at different scales
Kotin, Robert M.
Journal of virological methods
The conventional methods for producing recombinant adeno-associated virus (rAAV) rely on transient transfection of adherent mammalian cells. To gain acceptance and achieve current good manufacturing process (cGMP) compliance, clinical grade rAAV production process should have the following qualities: simplicity, consistency, cost effectiveness, and scalability. Currently, the only viable method for producing rAAV in large-scale, e.g.≥1016 particles per production run, utilizes Baculovirus Expression Vectors (BEVs) and insect cells suspension cultures. The previously described rAAV production in 40 L culture using a stirred tank bioreactor requires special conditions for implementation and operation not available in all laboratories. Alternatives to producing rAAV in stirred-tank bioreactors are single-use, disposable bioreactors, e.g. Wave™. The disposable bags are purchased pre-sterilized thereby eliminating the need for end-user sterilization and also avoiding cleaning steps between production runs thus facilitating the production process. In this study, rAAV production in stirred tank and Wave™ bioreactors was compared. The working volumes were 10 L and 40 L for the stirred tank bioreactors and 5 L and 20 L for the Wave™ bioreactors. Comparable yields of rAAV, ~2e+13 particles per liter of cell culture were obtained in all volumes and configurations. These results demonstrate that producing rAAV in large scale using BEVs is reproducible, scalable, and independent of the bioreactor configuration. Keywords: adeno-associated vectors; large-scale production; stirred tank bioreactor; wave bioreactor; gene therapy.
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