Development of TetraVecta™ Lentiviral Vectors: New Technology To Improve Their Production, Quality, Safety, Capacity and Utility.
J Wright(1) B Alberts(1) C Nogueira(1) B Caratti(1) K A Mitrophanous(1) N G Clarkson(1) D C Farley(1)
1:Oxford Biomedica (UK) Limited, Windrush Court, Transport Way, Oxford, OX4 6LT, UK
As clinical use of Lentiviral vectors (LVVs) expands there is a need to continually improve vector production, quality, safety, capacity and potency. Here, we describe new technologies that improve these properties and underpin a new class of TetraVecta™ LVV genomes.
Our TetraVecta™ LVVs come in two core designs:  Rev-dependent ‘2KO-LVVs’ and  Rev-independent ‘MaxPax’ LVVs. Both platforms harbour a modification that inactivates the major splice donor (MSD) site within the packaging sequence. This modification eliminates aberrant splicing therefore simplifies full length vRNA production.
Since MSD-inactivation alone leads to a reduction in LVV out-put titres, we have engineered different solutions to reverse this effect. 2KO-LVVs titres are maximised by the in-production co-expression of a modified U1 snRNA targeted to the LV packaging region. For MaxPax-LVVs, the attenuating effects of the MSD-inactivation are abrogated using a programmed splice event that stabilises the vRNA without contributing to overall vRNA length. Removal of RRE and most of the gag sequences allow MaxPax-LVVs to package ~1kb of extra transgene sequence.
Our TetraVecta™ LVVs also employ a ‘sequence-upgraded polyA’ or supA LTRs, providing improved transcriptional insulation for the integrated LVV in patient cells over current vectors. SupA-LTRs reduce transcriptional read-in to the cassette from adjacent chromatin by up to 50-fold whilst increasing transgene expression.
Finally, these features synergise with the ‘Transgene Repression In vector Production’ system (TRiP system™), which improves LV production and safety by reducing transgene expression during vector production as well as incorporation of the transgene protein into particles.