Efficient protection of microorganisms for delivery to the intestinal tract by cellulose sulphate encapsulation.

BACKGROUND: Gut microbiota in humans and animals play an important role in health, aiding in digestion, regulation of the immune system and protection against pathogens. Changes or imbalances in the gut microbiota (dysbiosis) have been linked to a variety of local and systemic diseases, and there is growing evidence that restoring the balance of the microbiota by delivery of probiotic microorganisms can improve health. However, orally delivered probiotic microorganisms must survive transit through lethal highly acid conditions of the stomach and bile salts in the small intestine. Current methods to protect probiotic microorganisms are still not effective enough. RESULTS: We have developed a cell encapsulation technology based on the natural polymer, cellulose sulphate (CS), that protects members of the microbiota from stomach acid and bile. Here we show that six commonly used probiotic strains (5 bacteria and 1 yeast) can be encapsulated within CS microspheres. These encapsulated strains survive low pH in vitro for at least 4 h without appreciable loss in viability as compared to their respective non-encapsulated counterparts. They also survive subsequent exposure to bile. The CS microspheres can be digested by cellulase at concentrations found in the human intestine, indicating one mechanism of release. Studies in mice that were fed CS encapsulated autofluorescing, commensal E. coli demonstrated release and colonization of the intestinal tract. CONCLUSION: Taken together, the data suggests that CS microencapsulation can protect bacteria and yeasts from viability losses due to stomach acid, allowing the use of lower oral doses of probiotics and microbiota, whilst ensuring good intestinal delivery and release.

The 5'-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation.

In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5'-untranslated region (5'-UTR) of the mouse mammary tumor virus (MMTV). The 5'-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5'-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5'-UTR was resistant to the addition of m(7)GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5'-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.

Magnetic field-controlled gene expression in encapsulated cells.

Cell and gene therapies have an enormous range of potential applications, but as for most other therapies, dosing is a critical issue, which makes regulated gene expression a prerequisite for advanced strategies. Several inducible expression systems have been established, which mainly rely on small molecules as inducers, such as hormones or antibiotics. The application of these inducers is difficult to control and the effects on gene regulation are slow. Here we describe a novel system for induction of gene expression in encapsulated cells. This involves the modification of cells to express potential therapeutic genes under the control of a heat inducible promoter and the co-encapsulation of these cells with magnetic nanoparticles. These nanoparticles produce heat when subjected to an alternating magnetic field; the elevated temperatures in the capsules then induce gene expression. In the present study we define the parameters of such systems and provide proof-of-principle using reporter gene constructs. The fine-tuned heating of nanoparticles in the magnetic field allows regulation of gene expression from the outside over a broad range and within short time. Such a system has great potential for advancement of cell and gene therapy approaches.