Using viral vectors as gene transfer tools (Cell Biology and Toxicology Special Issue: ETCS-UK 1 day meeting on genetic manipulation of cells)

In recent years, the development of powerful viral gene transfer techniques has greatly facilitated the study of gene function. This review summarises some of the viral delivery systems routinely used to mediate gene transfer into cell lines, primary cell cultures and in whole animal models. The systems described were originally discussed at a 1-day European Tissue Culture Society (ETCS-UK) workshop that was held at University College London on 1st April 2009. Recombinant-deficient viral vectors (viruses that are no longer able to replicate) are used to transduce dividing and post-mitotic cells, and they have been optimised to mediate regulatable, powerful, long-term and cell-specific expression. Hence, viral systems have become very widely used, especially in the field of neurobiology. This review introduces the main categories of viral vectors, focusing on their initial development and highlighting modifications and improvements made since their introduction. In particular, the use of specific promoters to restrict expression, translational enhancers and regulatory elements to boost expression from a single virion and the development of regulatable systems is described

Gene therapy for monogenic liver diseases: clinical successes, current challenges and future prospects

Over the last decade, pioneering liver-directed gene therapy trials for haemophilia B have achieved sustained clinical improvement after a single systemic injection of adeno-associated virus (AAV) derived vectors encoding the human factor IX cDNA. These trials demonstrate the potential of AAV technology to provide long-lasting clinical benefit in the treatment of monogenic liver disorders. Indeed, with more than ten ongoing or planned clinical trials for haemophilia A and B and dozens of trials planned for other inherited genetic/metabolic liver diseases, clinical translation is expanding rapidly. Gene therapy is likely to become an option for routine care of a subset of severe inherited genetic/metabolic liver diseases in the relatively near term. In this review, we aim to summarise the milestones in the development of gene therapy, present the different vector tools and their clinical applications for liver-directed gene therapy. AAV-derived vectors are emerging as the leading candidates for clinical translation of gene delivery to the liver. Therefore, we focus on clinical applications of AAV vectors in providing the most recent update on clinical outcomes of completed and ongoing gene therapy trials and comment on the current challenges that the field is facing for large-scale clinical translation. There is clearly an urgent need for more efficient therapies in many severe monogenic liver disorders, which will require careful risk-benefit analysis for each indication, especially in paediatrics

CAP Technology: Production of Influenza Vaccine in Human Amniocytes

The pressure to innovate in cell-based production systems is weighting particularly strong on the manufacturers of vaccines, as the currently used manufacturing systems do encounter severe problems. In the case of currently used egg-based vaccine manufacturing technology an average production campaign of e.g. influenza vaccines takes up to 6 months. In contrast to the egg-based production technology, cell culture-based systems show the potential to drastically shorten production cycles and improve quality of the vaccine regarding e.g. glycosylation of immunogenic antigens and risk of animal contaminants. CEVECs Amniocyte Production (CAP) cell technology is a versatile production platform to generate difficult-to-express and manufacture therapeutic proteins. The generation and development of the CAP human cell system has been fully documented according to all relevant regulatory standards. During the past years CAP cells have been broadly used for production of complex proteins and antibodies and thus have proved its strong potential as new technology platform for proteins in high yields and with authentic human glycosylation pattern. Addressing the needs of vaccine manufacturers, we have tested the suitability of CAP cells for the production of influenza vaccines. CAP cells growing in suspension in serum-free medium were evaluated for their potential as host cells for different human and animal influenza strains. The studies included extracellular metabolite concentrations during growth and virus production in different cultivation systems, test of different commercial serum-free media and evaluation of process conditions (trypsin concentration, multiplicity of infection, media feeding). In addition, time-course of infection and virus adaptation was characterized, and virus yields obtained with CAP cells were compared to those with MDCK cells. Favorable robust process parameters and high virus yields obtained with different influenza strains demonstrated that CAP cells are very promising candidates for large-scale manufacturing of vaccines in serum-free medium

CAP Technology: Production of Influenza Vaccine in Human Amniocytes

The pressure to innovate in cell-based production systems is weighting particularly strong on the manufacturers of vaccines, as the currently used manufacturing systems do encounter severe problems. In the case of currently used egg-based vaccine manufacturing technology an average production campaign of e.g. influenza vaccines takes up to 6 months. In contrast to the egg-based production technology, cell culture-based systems show the potential to drastically shorten production cycles and improve quality of the vaccine regarding e.g. glycosylation of immunogenic antigens and risk of animal contaminants. CEVECs Amniocyte Production (CAP) cell technology is a versatile production platform to generate difficult-to-express and manufacture therapeutic proteins. The generation and development of the CAP human cell system has been fully documented according to all relevant regulatory standards. During the past years CAP cells have been broadly used for production of complex proteins and antibodies and thus have proved its strong potential as new technology platform for proteins in high yields and with authentic human glycosylation pattern. Addressing the needs of vaccine manufacturers, we have tested the suitability of CAP cells for the production of influenza vaccines. CAP cells growing in suspension in serum-free medium were evaluated for their potential as host cells for different human and animal influenza strains. The studies included extracellular metabolite concentrations during growth and virus production in different cultivation systems, test of different commercial serum-free media and evaluation of process conditions (trypsin concentration, multiplicity of infection, media feeding). In addition, time-course of infection and virus adaptation was characterized, and virus yields obtained with CAP cells were compared to those with MDCK cells. Favorable robust process parameters and high virus yields obtained with different influenza strains demonstrated that CAP cells are very promising candidates for large-scale manufacturing of vaccines in serum-free medium