PROJECT SUMMARY
Rising expense associated with stability and delivery are significant obstacles for using therapeutic proteins to
treat diseases. To resolve these issues, we propose to have the system that is making the therapeutic protein
also be the direct delivery system. Delivering proteins directly from an expression system to a target tissue has
the advantage of bypassing purification and eliminating stability issues during storage. Improvements to protein
expression and secretion in Pichia pastoris make this yeast a prime candidate for this purpose. We have
previously reported on P. pastoris’s ability to grow with cultured mammalian cells and secrete basic fibroblast
growth factor to influence fibroblast growth. We have since tested P. pastoris’s ability to live in the gastrointestinal
(GI) tract of C57BL/6J mice. The preliminary results showed that P. pastoris DNA was detectible in the murine
feces 24 hours after the last treatment, while green fluorescent protein secreted by this strain of P. pastoris was
detectible 72 hours after the last treatment. The fecal samples from P. pastoris-treated mice yielded no
measurable levels of calprotectin, a biomarker of inflammation. Histological analysis of the small intestine,
cecum, and colon showed no obvious signs of inflammation while enzyme-linked immunosorbent assay analysis
of the GI tissues detected no increase in tumor necrosis factor alpha or interferon gamma. Furthermore, P.
pastoris-treated mice gained similar weight to control mice and exhibited no noticeable change in behavior. In
continuing with studies in animals, the goals of this proposal are to extend the duration of which P. pastoris can
remain in the murine gut and to provide proof of concept that P. pastoris is capable of surviving and delivering
recombinant proteins in the GI tract. Specific Aim 1 will determine if increasing dose and duration of treatment
will allow P. pastoris to remain in the gut for a longer time. To further enhance P. pastoris survival, we will
engineer a new strain of P. pastoris that will have affinity to mucin, an abundant protein in the mucus layer which
lines the entire GI tract. The effects of these treatment strategies on the inflammatory response in the murine
gut will also be assessed. In Specific Aim 2, we will use a hyper-lactose fed mouse model to determine if P.
pastoris can secrete recombinant proteins into the gut. A diet of 70% lactose causes adult mice to lose weight
due to an inability to digest lactose. Since lactase hydrolyzes lactose, a b-galactosidase (lactase)- secreting P.
pastoris strain that lives in the GI tract would be expected to enhance lactose digestion. Prevention of weight
loss in hyper-lactose fed mice would indicate that P. pastoris is secreting active lactase, suggesting that the
yeast has potential of secreting other heterologous proteins in the gut of mice as well. These studies establish a
foundational basis, upon which proteins made by P. pastoris can be directly administered to the GI tract of
animals as therapeutic agents for treating diseases.
