|IL-2 and HIV | type I IFN and arthritis | recombinant cytokine research|
» recombinant cytokines:
» IL-2 and HIV:
» type I IFN and arthritis:
» recombinant cytokine research:
recombinant IL-2 and tissue-specific expressionInterleukin-2 expression in plants
Another medically important cytokine that has enjoyed research focus is interleukin-12 (IL-12). This molecule sometimes works in cooperation with IL-2 and is a pro-inflammatory cytokine that mostly acts on lymphocytes (B and T cells) and NK (natural killer) cells[1,2]. It has been shown in early clinical tests to have strong potential in treating cancers by inhibiting angiogenesis and tumorigenesis. It has been co-administered as an adjuvant to induce protective immunity against microbials and viruses, significantly HIV. IL-12 has been expressed in tobacco and tomato plant systems in an ingenious way. Human IL-12 is naturally a heterodimer of two α- and β-chains, which are encoded by two disparate genes. The two chains only associate after translation and are only functional after association. High β-chain concentrations lead to β-homodimers, which repress the natural heterodimer's activity. Hence, the expression of precisely corresponding subunits is of utmost importance in transgenic systems for efficiency and product usability. To circumvent this problem, recombinant IL-12 has been expressed instead as a single-chain fusion polypeptide, in which the α- and β-chains are kept together by a linkage sequence[2,3]. Bioactivity of the recombinant molecule has so far proven to be the same as the natural one, by the induction of interferon-γ (IFN-γ) secretion in NK cells.
Difference between plant systems
Studies involving transgenic IL-12 have shown that although tobacco systems do not have high expression and generate toxic alkaloids in their crude extracts, tomatoes demonstrate a much higher, and more usable level of expression in both their fruit and leaves. Oddly, both tobacco and tomato studies employed the standard CaMV 35S promoter, so the difference in expression is either due to the nature of the plant itself or the transgene (the tobacco and tomato studies used recombinant human and mouse IL-12 genes, respectively). These issues of inconsistent expression should be better examined before adapting the process industrially. So far, the evidence is that the tomato crude extracts contain already biologically active IL-12. Since tomato is edible, this presents the possibility of mass-accessibility to prophylactic and therapeutic treatment of IL-12 through simple oral ingestion.
Similar studies of IFN-α expression through transgenic potato systems have also brought up this point. Potato extracts with low transgenic IFN-α concentrations were administered orally to mice, and was shown to confer protection against bacterial infection at a level comparable to a much higher amount of purified natural IFN-α8. Clearly, the system the authors used provided some sort of additional biochemical benefit. Intranasal delivery has also been suggested for IL-12, since it is known to improve mucosal immunity. This route also reduces toxicity. Again, in vivo experiments must be further conducted.
Plants provide natural storage sites in their seeds. Studies have managed to express human granulocyte macrophage colony stimulating factor (hGM-CSF) in tobacco plants in a tissue-specific manner using a rice endosperm-specific glutelin promoter. The hGM-CSF was expressed up to 0.03% of the total soluble protein of the seed extracts, or about 287ng per milligram of extract. Bioassays showed that the protein was functional by inducing growth in TF-1 cells. However, plant-animal glycosylation differences that were observed in the earlier tomato IL-12 study were present in the recombinant hGM-CSF products as well[2,6]. hGM-CSF's function is not dependent on its glycosylation, but since no plant-made cytokines have made it to food safety trials, it is difficult to say whether this will be a setback. In any event, this tissue-specific expression is a gladdening success, for it suggests cytokines can be administered in a simple stable way, so that patients receive a high concentration in the form of a so-called "seed pill". Seeds, unlike extracts, or fruits, are hardier and last longer, surviving transport and long periods of storage.
references1) E.N. Fish. Personal communication on "Cytokines". February 2006.
2) Gutierrez-Ortega A, Avila-Moreno F, Saucedo-Arias LJ, Sanchez-Torres C, Gomez-Lim MA: Expression of a single-chain human interleukin-12 gene in transgenic tobacco plants and functional studies. Biotechnol Bioeng 2004, 85: 734-40.
3) Gutierrez-Ortega A, Sandoval-Montes C, de Olivera-Flores TJ, Santos-Argumedo L, Gomez-Lim MA: Expression of functional interleukin-12 from mouse in transgenic tomato plants. Transgenic Res 2005, 14: 877-85.
4) Ohya K, Matsumura T, Itchoda N, Ohashi K, Onuma M, Sugimoto C: Ability of orally administered IFN-alpha-containing transgenic potato extracts to inhibit Listeria monocytogenes Infection. J Interferon Cytokine Res. 2005, 25: 459-66.
5) Ohya K, Matsumura T, Ohashi K, Onuma M, Sugimoto C. Expression of two subtypes of human IFN-alpha in transgenic potato plants. J Interferon Cytokine Res 2001, 21: 595-602.
6) Sardana RK, Alli Z, Dudani A, Tackaberry E, Panahi M, Narayanan M, Ganz P, Altosaar I: Biological activity of human granulocyte-macrophage colony stimulating factor is maintained in a fusion with seed glutelin peptide. Transgenic Res 2002, 11: 521-31.