We're engineering a genetic circuit in yeast to accelerate human wound-healing with the goal of preventing dangerous infection which is often exacerbated in chronic or slow-healing wounds. We hope to accomplish this by locally expressing a set of growth factors associated with accelerated healing in a time-dependent fashion by microorganisms at the wound site.
Wound contamination is a double-edged sword. Virtually all wounds (~98%) are contaminated by aerobic organisms such as yeast and bacteria. The vast majority of these are benign, and many species actually aid the wound-healing process. On the other hand, the longer a wound remains unhealed, the greater the probability of dangerous infection by harmful pathogens. These dangerous infections can cause serious illness and even be life-threatening. In other words, microorganisms living at a wound-site are not necessarily bad, and often do good insofar as they accelerate the healing process or prevent serious infection.
The wound-healing process itself is a very complicated set of interdependent processes, but one bottleneck that can be alleviated is angiogenesis, the re-growth of vasculature. This can be accelerated by sequentially expressing vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) in a time-dependent fashion in response to hypoxia-inducible factor (HIF-1). When expressed locally at appropriate levels, this has the potential to appreciably accelerate rates of wound-healing with few side-effects. The challenge is to design a genetic circuit that achieves optimal rates of HIF-linked growth factor expression without over-shooting target expression because over-expression of VEGF can have very serious side-effects.
After consulting our advisers, we decided to use a yeast chassis to propagate our circuit. Of the possible host organisms (yeast and bacteria), yeast are better suited to this application because they possess the complicated biomolecular machinery required to assemble the complex mammalian growth factors in question. We also had readier access toresources to effectively undertake yeast lab protocol. We understand that future applications of our circuit will most probably not utilize yeast as the shuttle organism, due to complications it may pose when interacting with mammalian immune systems. Therefore future applications of our project would ideally utilize more complex mammalian cells (such as monocytes) that readily integrate within the human body (especially if extracted from the patient host), avoiding possible complications from an immune response.
Below is a diagram of our final genetic circuit with a brief description.
Figure 1. The circuit shows the expression of VEGF and LuxR governed by a hypoxia-inducible-factor-1 (HIF-1) promoter. The LuxR/LuxI quorum sensing system was selected from Vibrio fischeri as an adequate mechanism to communicate between both components of the circuit. Yeast also contains an acyl-homoserine lactone homologue to ensure that the system works effectively. The LuxR in conjunction with the acyl-homoserine lactone produced by constitutively expressed LuxI, forms a dimer which activates the LuxR promoter, thus driving the expression of the PDGF growth factor and a negative control siRNA. THe siRNA component will be complementary to a select random sequence preceding a kozak ribosomal binding site. The random sequence ensures specificity within our circuit (thus not inhibiting host cell machinery) while also enabling a more modular circuit (in case the inhibited gene must be replaced.) The siRNA offers negative control at the translational level, thus ceasing the expression of VEGF, while not inhibiting the expression of LuxR which ultimately drives the expression of PDGF. The LuxR promter must be tuned to match a threshold concentration of LuxR, corresponding to the necessary expression levels of VEGF needed to activate angiogenic processes.
See Virginia 2011's project wiki for more details.
From left to right: Yong Wu (ChemE 2012), Jackie Niu (BME 2010), Josh Fass (BME 2014), Arjun Artheya (Psychology 2012), Yanzhi Yang (ChemE 2014)