Biosynthesis Of Pharmaceutical Artificial-microRNA Using Plant

Background:Bioreactors are widely used in biopharmacy and the main expression systems include bacteria, yeasts, insect cells, mammalian cells and plants. Due to the outstanding features such as easy operation, environmental safety, low cost and pathogen-free, plant bioreactors have gained a lot of attention and some biopharmaceutical proteic products synthesized by plants have been available on the current market. However, the application of these plant bioreactors is still limited ascribed to the low expression level and the low homology of human proteins of their proteic products.Currently, the polypeptides or proteins are the major biopharmaceutical products. But the structure complexity of such products results in the difficulty of the targeting and and binding. Hence, nucleic acid drugs, especially small RNAs used in gene therapy become more attractive.RNA interference (RNAi) refers to two categories of these small RNAs ---siRNAs (short interfering RNAs) and miRNAs (microRNAs). The siRNAs are exogenous and the miRNAs are endogenous. In all cases, the identities of the genes to be silenced are specified by miRNA and siRNA, which recognizes each target by Watson-Crick base pairing. They both target genes by cleavage or translational inhibition of their mRNA. It plays an important role in basic activities of cells. In comparison with conventional treatment, RNAi has a unique advantage in gene therapy:high efficiency and specificity, low toxicity, high transmissibility.Currently, small RNAs used in RNAi therapeutic mainly come from chemical synthesis. Due to their instability and readily degradation, chemically synthesized siRNAs need additional chemical modification, which results in high cost. Besides, another major obstacle during RNAi therapeutic is the siRNAs delivery in vivo, i.e. how the siRNAs can be delivered to the target tissue or the organ lesion. A recent report showed that exogenous plant/food-derived miRNAs were present in the sera of humans or plant-feeding animals. Among of them miR168a could inhibit LDLRAP1 expression in liver. It suggests that exogenous plant miRNAs in food might regulate the expression of target genes in mammals.Purpose and Meaning:Based on the above studies, we proposed that the therapeutic small RNAs artificially expressed through plant could be uptaken by humans or animals to exert RNAi function. This study will not only solve the problems existing during RNAi therapeutic mentioned-above, but also enlarge the application of the plant bioreactor.Methods and Results:My thesis focused on the small interfering RNAs which against hepatitis C and type-2 diabete expressed in plant. Small interfering RNA sequences complement to HCV viral genome or PGCla mRNA were designed and transformed into lettuce via Agrobacterium-mediated transgenic technology. The expression of artificial miRNAs was confirmed by RT-PCR and Northern blot. More animal experiments or clinical trials are required to further validate the exact physiological and therapeutic functions of artificial miRNA.