RESUMO
Plants have been proposed as an attractive alternative for pharmaceutical protein production to current mammalian or microbial cell-based systems. Eukaryotic protein processing coupled with reduced production costs and low risk for mammalian pathogen contamination and other impurities have led many to predict that agricultural systems may offer the next wave for pharmaceutical product production. However, for this to become a reality, the quality of products produced at a relevant scale must equal or exceed the predetermined release criteria of identity, purity, potency and safety as required by pharmaceutical regulatory agencies. In this article, the ability of transient plant virus expression systems to produce a wide range of products at high purity and activity is reviewed. The production of different recombinant proteins is described along with comparisons with established standards, including high purity, specific activity and promising preclinical outcomes. Adaptation of transient plant virus systems to large-scale manufacturing formats required development of virus particle and Agrobacterium inoculation methods. One transient plant system case study illustrates the properties of greenhouse and field-produced recombinant aprotinin compared with an US Food and Drug Administration-approved pharmaceutical product and found them to be highly comparable in all properties evaluated. A second transient plant system case study demonstrates a fully functional monoclonal antibody conforming to release specifications. In conclusion, the production capacity of large quantities of recombinant protein offered by transient plant expression systems, coupled with robust downstream purification approaches, offers a promising solution to recombinant protein production that compares favourably to cell-based systems in scale, cost and quality.
Assuntos
Anticorpos Monoclonais/biossíntese , Aprotinina/biossíntese , Engenharia Genética/métodos , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/biossíntese , Anticorpos Monoclonais/imunologia , Aprotinina/imunologia , Vírus de Plantas , Plantas Geneticamente Modificadas/imunologia , Proteínas Recombinantes/imunologia , RhizobiumRESUMO
Historically, the study of plant viruses has contributed greatly to the elucidation of eukaryotic biology. Recently, concurrent with the development of viruses into expression vectors, the biotechnology industry has developed an increasing number of disease therapies utilizing recombinant proteins. Plant virus vectors are viewed as a viable option for recombinant protein production. Employing pathogens in the process of creating added value to agriculture is, in effect, making an ally from an enemy. This review discusses the development and use of viruses as expression vectors, with special emphasis on (+) strand RNA virus systems. Further, the use of virus expression vectors in large-scale agricultural settings to produce recombinant proteins is described, and the technical challenges that need to be addressed by agriculturists and molecular virologists to fully realize the potential of this latest evolution of plant science are outlined.
Assuntos
Agricultura/métodos , Vírus de Plantas/metabolismo , Plantas/virologia , Comovirus/genética , Comovirus/metabolismo , Regulação da Expressão Gênica de Plantas , Regulação Viral da Expressão Gênica , Vetores Genéticos/genética , Vírus de Plantas/genética , Plantas/genética , Vírus Eruptivo da Ameixa/genética , Vírus Eruptivo da Ameixa/metabolismo , Potexvirus/genética , Potexvirus/metabolismo , Vírus de RNA/genética , Vírus de RNA/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vírus do Mosaico do Tabaco/genética , Vírus do Mosaico do Tabaco/metabolismo , Tombusvirus/genética , Tombusvirus/metabolismo , VacinasRESUMO
Lysosomal acid lipase (LAL) is an essential enzyme that hydrolyzes triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes. Genetic LAL mutations lead to Wolman disease (WD) and cholesteryl ester storage disease (CESD). An LAL-null (lal(-/-)) mouse model resembles human WD/CESD with storage of CEs and TGs in multiple organs. Human LAL (hLAL) was expressed in Nicotiana benthamiana using the GENEWARE expression system (G-hLAL). Purified G-hLAL showed mannose receptor-dependent uptake into macrophage cell lines (J774E). Intraperitoneal injection of G-hLAL produced peak activities in plasma at 60 min and in the liver and spleen at 240 min. The t(1/2) values were: approximately 90 min (plasma), approximately 14 h (liver), and approximately 32 h (spleen), with return to baseline by approximately 150 h in liver and approximately 200 h in spleen. Ten injections of G-hLAL (every 3 days) into lal(-/-) mice produced normalization of hepatic color, decreases in hepatic cholesterol and TG contents, and diminished foamy macrophages in liver, spleen, and intestinal villi. All injected lal(-/-) mice developed anti-hLAL protein antibodies, but suffered no adverse events. These studies demonstrate the feasibility of using plant-expressed, recombinant hLAL for the enzyme therapy of human WD/CESD with general implications for other lysosomal storage diseases.