Improved production and quality of lentiviral vectors by major-splice-donor mutation and co-expression of a novel U1 snRNA-based enhancer

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 system™ (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 TetraVecta™ 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 system™, and represent a further development in the utility and safety of LVs.