Genetic engineering of a Lemna isoleucine auxotroph.

Lemna, a member of the Lemnaceae or duckweed family, is a small aquatic plant that can be quickly transformed to produce recombinant proteins in a contained and controlled bioprocessing environment. The containment capability of Lemna has been further improved with the creation of an auxotroph platform that requires isoleucine supplementation for survival of transformed plant lines. Using an RNAi based approach, threonine deaminase (TD) expression was targeted and thus resulted in dramatically reduced expression of this key enzyme in the isoleucine biosynthesis pathway. Auxotrophic plants expressing RNAi for TD were generated in the presence of isoleucine and selected based on their inability to propagate without isoleucine supplementation. TD transcripts isolated from the superior auxotroph lines were shown to be less than 10% of wild type level and thus confirmed the auxotroph phenotype to be derived from the specific knock down of TD expression. When grown under optimal conditions with appropriate isoleucine supplementation, biomass accumulation of the auxotroph lines was equivalent to that of wild type plants. To demonstrate the application of this system for production of recombinant proteins, an avian influenza H5N1 hemagglutinin (HA) protein was expressed in the isoleucine auxotroph platform. The successful expression of H5N1 HA vaccine antigen, in the isoleucine auxotroph background demonstrates the applicability of using an auxotroph to express biotherapeutics and vaccines in a highly contained expression system.

Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor.

N-glycosylation is critical to the function of monoclonal antibodies (mAbs) and distinguishes various systems used for their production. We expressed human mAbs in the small aquatic plant Lemna minor, which offers several advantages for manufacturing therapeutic proteins free of zoonotic pathogens. Glycosylation of a mAb against human CD30 was optimized by co-expressing the heavy and light chains of the mAb with an RNA interference construct targeting expression of the endogenous alpha-1,3-fucosyltransferase and beta-1,2-xylosyltransferase genes. The resultant mAbs contained a single major N-glycan species without detectable plant-specific N-glycans and had better antibody-dependent cell-mediated cytotoxicity and effector cell receptor binding activities than mAbs expressed in cultured Chinese hamster ovary (CHO) cells.

Geminivirus-based vectors for gene silencing in Arabidopsis.

Gene silencing, or RNA interference, is a powerful tool for elucidating gene function in Caenorhabditis elegans and Drosophila melanogaster. The vast genetic, developmental and sequence information available for Arabidopsis thaliana makes this an attractive organism in which to develop reliable gene-silencing tools for the plant world. We have developed a system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) that allows silencing of endogenous genes singly or in combinations in Arabidopsis. Two vectors were tested: a gene-replacement vector derived from the A component; and an insertion vector derived from the B component. Extensive silencing was produced in new growth from the A component vectors, while only minimal silencing and symptoms were seen in the B component vector. Two endogenous genes were silenced simultaneously from the A component vector and silencing of the genes was maintained throughout new growth. Because the CbLCV vectors are DNA vectors they can be inoculated directly from plasmid DNA. Introduction of these vectors into intact plants bypasses transformation and extends the kinds of silencing studies that can be carried out in Arabidopsis.