An Enterovirus-Like RNA Construct for Colon Cancer Suicide Gene Therapy

Background:

In gene therapy, the use of RNA molecules as therapeutic agents has shown advantages over plasmid DNA, including higher levels of safety. However, transient nature of RNA has been a major obstacle in application of RNA in gene therapy.

Methods:

Here, we used the internal ribosomal entry site of encephalomyocarditis virus and the 3’ non-translated region of Poliovirus to design an enterovirus-like RNA for the expression of a reporter gene (enhanced green fluorescent protein) and a suicide gene (thymidine kinase of herpes simplex virus). The expression of these genes was evaluated by flow cytometry and cytotoxicity assay in human colorectal adenocarcinoma cell line (SW480). We then armed RNA molecules with a target sequence for hsa-miR-143 to regulate their expression by microRNA (miRNA) mimics.

Results:

The results showed effective expression of both genes by Entrovirus-like RNA constructs. The data also showed that the restoration of hsa-miR-143 expression in SW480 leads to a significant translation repression of the introduced reporter and suicide genes.

Conclusion:

Collectively, our data suggest the potential use of Entrovirus-like RNA molecules in suicide gene therapy. Additionally, as a consequence of the possible downregulated miRNA expression in cancerous tissues, a decreased expression of gene therapy constructs armed with target sequences for such miRNA in cancer tissue is expected.Key Words: Thymidine kinase, Polio 3’NCR, miR-143     

Lipid oxidation and amylopectin molecular weight changes occurring during storage of extruded starch samples

Using thermomechanical extrusion, waxy maize starch and 4% (w/w) lipid were formed into strips. The lipids used were free fatty acids (mostly linoleic) and antioxidant stripped sunflower oil (either previously oxidised or fresh, with and without copper ions). By altering their water content it was possible to store, at the same temperature, sample strips in the glassy and rubbery states. Lipid oxidation was monitored by determining hexanal in the headspace above all stored samples. The molecular weights of the amylopectin in the samples of extruded products (dissolved in dimethylsulfoxide, precipitated and redissolved using a pressure cell) were determined by asymmetric flow-field-flow fractionation, analytical ultracentrifugation and light scattering. The initial rate of hexanal generation was higher in samples stored in the glassy state compared with those in the rubbery state. Waxy maize and extruded samples, at the start of the storage period and after 42 days, were used to establish the molecular weight of the amylopectin. A significant fall (a decrease of 40%) in molecular weight was found on storage of samples containing sunflower oil and copper ions and those containing free fatty acids, irrespective of the method used for molecular weight determination.