Abstract
A key challenge in the field of therapeutic viral vector/vaccine manufacturing is maximizing production. For most vector platforms, the ‘benchmark’ vector titres are achieved with inert reporter genes. However, expression of therapeutic transgenes can often adversely affect vector titres due to biological effects on cell metabolism and/or on the vector virion itself. Here, we exemplify the novel ‘Transgene Repression In vector Production’ (TRiP) system for the production of both RNA- and DNA-based viral vectors. The TRiP system utilizes a translational block of one or more transgenes by employing the bacterial tryptophan RNA-binding attenuation protein (TRAP), which binds its target RNA sequence close to the transgene initiation codon. We report enhancement of titres of lentiviral vectors expressing Cyclo-oxygenase-2 by 600-fold, and adenoviral vectors expressing the pro-apoptotic gene Bax by >150,000-fold. The TRiP system is transgene-independent and will be a particularly useful platform in the clinical development of viral vectors expressing problematic transgenes.
Highlights
A key challenge in the field of therapeutic viral vector/vaccine manufacturing is maximizing production
We have developed the Transgene Repression In vector Production’ (TRiP) system for the universal production of DNA- and RNA-based viral vectors that encode transgenes whose activity is detrimental to vector titres
To create the mammalian cell-based TRiP system, we codon-optimized the sequence of tryptophan RNA-binding attenuation protein (TRAP) from Bacillus subtilis for human codon bias and generated a simple gene-expression cassette (Fig. 2a)
Summary
A key challenge in the field of therapeutic viral vector/vaccine manufacturing is maximizing production. Contemporary gene therapy vectors based on RNA viruses such as g-Retroviruses and Lentiviruses, and DNA viruses such as Adenovirus and Adeno-associated virus (AAV) have shown promise in a growing number of human disease indications. These include ex vivo modification of patient cells for hematological conditions, and in vivo treatment of ophthalmic, cardiovascular, neurodegenerative diseases and tumour therapy. A substantial body of research exists on the characterization of natural TRAP function, with a particular focus on use of in vitro techniques16–19 This has revealed an extremely high-affinity of TRAP for an optimal tbs [KAGNN]11 in the nanomolar range. This led others to apply this stable TRAP/tbs complex as a positive control in development of a screening tool for novel RNA-binding proteins in mammalian cells
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