Quantitative RNA Analysis and Roles of Viral RNA Silencing Suppressors in Transient Therapeutic Protein Production in Plant Tissues using a Viral Amplicon-Based System

Plant-based production of therapeutic proteins has become an attractive platform for large-scale manufacturing due to cost effectiveness, the capabilities of eukaryotic post-translational modification, and safety. Plant virus based expression systems are an effective system for transient expression in plants due to high levels of transgene expression resulting from viral replication. Sudarshana et al. have developed a regulated, chemically inducible viral based expression system for Agrobacterium tumefaciens -mediated transient expression in plants, referred to as CMViva ( Cucumber mosaic virus inducible viral amplicon). In the CMViva system, the plant codon optimized human alpha-1-antitrypsin (AAT) with rice alpha amylase 3D (RAmy3D) signal peptide sequences have replaced the open reading frame of the CMV coat protein. In this study, we use the CMViva system for production of recombinant alpha-1-antitrypsin (rAAT) in harvested leaves of wild-type Nicotiana benthamiana plants using the vacuum agroinfiltration method. The effect of transient co-expression of viral RNA silencing suppressors (VRSS) from various plant viruses was evaluated. Tomato bushy stunt virus (TBSV) P19, Turnip mosaic virus (TuMV) P1/HcPro and Grapevine leafroll-associated virus (GLRaV) P24, significantly improved functional rAAT level by 68 fold, 30-fold and 15-fold, respectively compared to without co-expression of VRSS at 6 days post-induction (dpi). In addition, the optimal concentrations of A. tumefaciens harboring binary plasmid containing TBSV p19 and TuMV p1/HcPro for co-expressions with the CMViva system were at an optical density (OD600nm) of 0.06 and 0.25, respectively. The bioprocessing conditions of rAAT expression using the CMViva system were also optimized. Using a 1-minute vacuum agroinfiltration with A. tumefaciens harboring pCMVia-SPAAT and pBIN-p19 at a concentration corresponding to OD600nm of 0.5 and 0.06, respectively, and a 1-minute vacuum induction with beta-estradiol at a concentration of 25 muM, the maximum functional and total rAAT levels at 4 days post-induction were 261+/-86 mg rAAT/kg FW and 280+/-153 mg rAAT/kg FW, respectively.;Furthermore, RNA kinetics of CMV ORFs, our target protein and p19 were measured by using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). The RNA levels of CMV ORFs and mRNA of AAT reached their maximum at 2 days after induction. The maximum RNA levels of AAT obtained from the CMViva system were much higher than the constitutive Cauliflower mosaic virus (CaMV) 35S system. For the study of rAAT production kinetics, the CMViva system increased the ratio of functional rAAT to total rAAT and increased the functional rAAT levels by 33-fold higher than the CaMV 35S system. This work gives an understanding of the molecular basis for transient protein production using the CMViva system and also confirms the advantages of viral-based expression system for of transient heterologous protein production in harvested plant tissue. Finally, this study demonstrates that transient, viral-based expression systems are a highly efficient platform for functional therapeutic protein production in plants.