(820d) Quantitative Intracellular Analysis of the Cucumber Mosaic Virus Chemically Inducible Viral Amplicon Expression System

Transient production of heterologous proteins in plant tissues is gaining importance as a commercially-relevant production strategy due to its inherent speed, scalability, and ability to produce complex glycoproteins.   Fast and efficient protein expression is essential for cost effective production of biologics in plants. Plant viral based expression systems provide rapid high-level expression through the viral replication mechanism.  In the novel chemically inducible viral amplicon expression system referred to as CMViva (Cucumber Mosaic Virus inducible viral amplicon) the open reading frame of the CMV coat protein is replaced with the plant codon optimized gene for the target product, which along with other modifications of cDNA of the CMV genome, ensure that infectious CMV virions are not generated. Genetically engineered Agrobacterium tumefaciens harboring the CMViva expression system is delivered into harvested Nicotiana benthamianaleaf tissue via vacuum agroinfiltration. A viral RNA gene silencing suppressor (GSS) driven by the CaMV 35S promoter is co-expressed to improve production of the target.  

In this study the target product is alpha-1-antitrypsin (AAT), a labile 52kD human glycoprotein.  People with AAT genetic disorder lack functional AAT in their bloodstream and lungs, leading to early onset emphysema which often requires weekly AAT replacement therapy.  Plasma derived AAT is currently the only FDA approved source, however due to production constraints, safety concerns and cost, alternative recombinant production methods are being investigated.

The absolute RNA copy numbers of CMV ORFs, AAT and GSS transcripts is determined using Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (real time qRT-PCR). The levels of CMV RNA 1, RNA 2, RNA 3, CMV 2b, AAT and GSS mRNAs are measured and compared under non-induced and induced conditions. The mRNA and protein expression levels of rAAT are also compared to those obtained with the constitutive CaMV 35S system. Using the CMViva system, the mRNA levels of the CMV ORFs, AAT and GSS mRNAs reached their maximum at 2 days after induction. The maximum mRNA levels of AAT obtained from the CMViva system were 8 fold higher than the constitutive 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 compared with the CaMV 35S system. These intracellular molecular level data support a conceptual model of the CMViva plant-based expression system.