The clinical success in using HIV-1 based Lentiviral vectors (LVs) to deliver therapeutic genes is built upon the safety features of 3rd generation LVs. However, the Cell and Gene Therapy industry as well as regulatory agencies are continually seeking improvements in product quality and safety. LV RNA genomes retain complex HIV-1 cis-acting sequences that govern production of unspliced/spliced vector (v)RNA, including the major splice donor (MSD) within the packaging signal. Substantial aberrant splicing from the MSD to strong/cryptic splice acceptors within transgene cassettes can occur during LV production. Such aberrant vRNAs encode the transgene, explaining why tissue-specific promoter-driven transgenes appear to be leaky during LV production. Additionally, the TRiP systemTM (Transgene Repression In vector Production), which represses translation of transgenes during production to enhance titres and/or omit the transgene protein from product, is less efficacious on these spliced vRNAs. We show that resultant spliced forms of vRNA can be detected in LV product and can be converted to episomal cDNA in target cells. MSD-mutation reduces LV titre in tat-independent systems, and therefore these limitations remain unsolved in contemporary 3rd generation LV platforms. We present a novel LV platform-referred to as the TetraVectaTM System-utilising optimised MSD-inactivating sequences, along with a new class of vRNA enhancer based on modified U1 snRNA. These enhancers, co-expressed during production, rescue the output titres of MSD-mutated LVs. MSD-mutated LVs do not generate spliced forms of vRNAs or the resultant episomal cDNAs, work optimally with the TRiP systemTM, and represent a further development in the utility and safety of LVs.

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.