By Cristina Nogueira, Ben M Alberts, Jordan Wright, Helen Maunder, Kyriacos A Mitrophanous, Nicholas G Clarkson and Daniel C Farley
Oxford Biomedica (OXB) have previously developed the TRiP System™ as a method for reducing the effect of transgene expression on out-put titres during Upstream phases, reducing potential immune responses to contaminating transgene protein in the final product, as well as improving feed quality for downstream purification. The TRiP system exploits the bacterial protein Tryptophan RNA-binding Attenuation Protein (TRAP) and a 55 nucleotide TRAP binding site (tbs) inserted upstream of the transgene open reading frame. Binding of TRAP protein to the tbs results in formation of stable RNP complex which inhibits translation initiation. The system therefore allows suppression of transgene expression during upstream processes, resulting in enhanced vector yield and purity, simplifying downstream processes.
This poster details improvements to the general tbs consensus sequence [KAGNN]x11, enhancements to the sequences within the untranslated region (UTR), and optimisation of tbs positioning in relation to the Kozak consensus sequence. The enhanced TRiP technology has been applied to chimeric antigen receptor (CAR) encoding lentiviral vectors (LVs), successfully reducing transgene expression in production cells resulting in improved yields.