Cereal crops as an ideal molecular pharming platform for human health

• Autores: Koreen Ramessar
• Directores de la Tesis: Paul Christou (dir. tes.), Teresa Capell Capell (codir. tes.)
• Lectura: En la Universitat de Lleida ( España ) en 2009
• Idioma: español
• Tribunal Calificador de la Tesis: Ignacio Romagosa Clariana (presid.), Ludovic Bassie (secret.), Eva Stoger (voc.), Julian K-C. Ma (voc.), Antonio Pedro Martin Muñoz (voc.)
• Materias:
  • Ciencias de la vida
    • Genética
      • Ingeniería genética
    • Inmunología
      • Anticuerpos
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• Resumen

  • Molecular pharming uses plants as efficient factories for producing recombinant pharmaceutical proteins, including antibodies, vaccines, blood proteins, and other molecules useful in diagnosis and therapy. Plants have many advantages compared with conventional production systems- they allow for low cost of production, are amenable to rapid and economical scale-up and are free of human pathogens. Plant production systems have the necessary biochemical machinery for folding, assembly and transport of complex recombinant proteins. Cereals, particularly maize, have well-established agronomic, harvesting, transport, storage and processing practices. Cereal seeds additionally offer high stability of proteins when dry and can be easily stored for long periods of time without loss of activity. Such crops are therefore an ideal, alternative production platform for the accumulation of functional, pharmaceutically important recombinant proteins (such as antibodies and antigens) for molecular pharming applications in human health and veterinary medicine. The focus of this thesis is the production of HIV-neutralizing antibodies and an autoantigen against type-1 diabetes, in maize seeds. High-level, endosperm-specific production of the HIV neutralizing 2G12 antibody in maize seed is demonstrated, with equal (if not superior) virus neutralization capacity when compared to the same molecule produced in mammalian cells. The glutamic acid decarboxylase (GAD65) autoantigen has also been successfully produced in maize seeds; further screening to identify high expressing seeds for use in mice oral tolerance studies continues. A second HIV neutralizing antibody (4E10) has been recently transformed into maize, and putative transgenic plants will soon be available for screening. Maximizing accumulation of recombinant protein(s) is an important goal in itself in molecular pharming and the key factors that affect recombinant protein yields in cereals are further evaluated within this thesis. Furthermore, many questions have been raised on how recombinant proteins are synthesized, transported and stored in plants, and how these affect recombinant protein quality in terms of protein folding and posttranslational modification and accumulation levels. For these reasons a mechanistic study of recombinant protein deposition in cereal endosperm formed an integral component of this thesis. Rice and maize were transformed with constructs encompassing the DsRed fluorescent marker protein to induce protein bodies in endosperm for subsequent proteomic and genomic studies. Monitoring of the fluorescent protein in vivo would allow sensitive macroscopic and microscopic detection of transgene activity in the endosperm, to monitor how protein synthesis, trafficking and deposition in seeds occur. These studies will aid in the development of more refined strategies for the optimal accumulation of valuable recombinant proteins for molecular pharming applications. Biosafety and regulatory issues relating to molecular pharming also form an important element of my thesis, as these constitute bottlenecks to the rapid development and commercialization of genetically engineered crops expressing plant-made pharmaceuticals, globally. The current national and international regulatory frameworks relating to the production, cultivation and trade of plants producing recombinant pharmaceuticals are discussed critically. The research program within this thesis therefore provides a holistic view of the science and political dimension of molecular pharming specifically targeted to the needs of developing countries.