Team:Penn/ProjectResults

What if you could combine spatial targeting and cellular targeting into the same therapeutic? This idea is unprecedented but would allow for precise targeting of specific cells within a specific area, leaving healthy tissue intact and keeping side effects to a minimum. Higher dose precision means more of the therapeutic would be used efficiently in the targeted area and the dependency on passive diffusion – and the uncertainties that comes with it – would be eliminated.

The 2012 Penn iGEM team has engineered a novel, modular platform for targeted therapeutics that employs simultaneous spatial and cellular targeting. We have achieved spatial (and temporal) targeting with a blue light-switchable transgene expression system and cellular targeting through display of an antibody-mimetic protein on the surface of E. coli for the first time. Our platform also enables more precise dose control in the targeted area through the length of blue-light exposure, which allows us to regulate effective levels of transgene expression.

As a proof of concept, we applied our system to the treatment of cancer, a disease in which spatial and cellular targeting are of utmost importance. We displayed a high-affinity antibody-mimetic protein that targets Human Epidermal Growth Factor Receptor 2 (HER2), a protein commonly overexpressed in cancer cells, especially in breast cancer tumors. We combined this cellular targeting with a light-activated cytotoxic protein delivery system to successfully target and kill breast cancer cells.

The strength in our platform lies in its modularity. Both the light-induced transgene expression system and the surface display system work and exist independently of each other on two compatible plasmids. These plasmids can be modified to meet the needs of any synthetic biologist.

Any gene of interest can be cloned into the light-induced transgene expression system and will then be expressed in a light-dependent and spatially controlled manner. Any targeting protein can be cloned into our surface display platform to allow cellular targeting against any desired biomarker. These modular plasmids may then be co-transformed together to create a bacteria capable of producing a desired therapeutic molecule in a spatially and cellularly targeted manner.

These modular components could also be extended into applications other than medical therapeutics, such as biocatalysis, manufacturing, and alternative energy.

Light Activated Gene Expression Module: Light is transduced into a chemical signal through the light sensor, which through a cascade of protein activation/repression results in the light-dependent downstream expression of a desired protein.	Surface Display Module: A target ligand is bound by a target binder displayed on the surface of the bacterial cell through the surface display module.

For our engineered bacterial therapeutic, we chose to target breast cancer as a proof of concept using a blue light gene expression system. We chose this specific blue-light inducible gene expression system because of the well-characterized nature of the parts and the high on:off ratio.

We also identified a well-characterized class of antibody mimetic proteins called designed ankryin repeat proteins, or DARPins. One DARPin in particular (H10-2-G3) was engineered to bind to the Human Epidermal Growth Factor 2 (HER2) at picomolar affinities. HER2 is overexpressed in breast cancer cells, and we had access to cell lines that overexpressed HER2 on their cell surface which we could use for binding assays.

In the future, we hope to be able to replace components in both modules of our system, allowing us to utilize different wavelengths of light to induce protein expression, target different sub populations of cells, and explore orthogonal uses for the surface display module unrelated to theraputic delivery. Further testing of our theraputic proof of concept in mice may also identify new strategies for reducing host immune system responses to the bacterial therapeutic.

Upon conception of this project, we realized that although hundreds of academic research projects and iGEM projects have been conducted in the realm of Health and Medicine, almost no engineered bacterial therapeutics have been brought to the clinic. We analyzed the hurdles and road ahead for bacterial synthetic biology-enabled therapeutics, compiling a thorough report with specific actions that iGEM teams in Health/Medicine can take to make their therapies more clinically tractable. This project directly informed our wet lab work, leading us to port our therapeutic system into a non-pathogenic, probiotic bacterial strain which is already used in human therapies today.

We hope our targeted therapeutic platform will allow other scientists and iGEM teams to target any cells they choose. In the near term, we are planning to test our cancer cell targeting/killing bacterial system in a mouse model and make a real impact on cancer research and therapy.