20.109(F10):Engineered Photoreceptors

This the the Silver Wiki Page.

Project Background
The photoreceptor is one of 5 key cell types in the eye. It works by sensing light and using a complex signal transduction pathway with the end result of releasing glutamate onto bipolar cells. This project will focus on developing an alternative signalling pathway for photoreceptors by using a similar route to the 2CS in the bacterial photography system in order to develop a photoreceptor that is not dependent upon specific properties of the pigmented epithelium, degeneration of which is the primary cause of AMD.

Structure and Objective
The primary challenge is developing a signal that leads to exocytosis of glutamate - specifically, bridging the gap between phosphorylation and exocytosis. This system will use the BPS pathway from the light sensing device up to an ompR analog. Hopefully this ompR analog can be used to phosphorylate something leading to exocytosis of glutamate. An ideal route would be a phosphorylation-gated glutamate channel. (Lit/references for this? - it doesn't seems to exist.) Another option is to introduce another component into the system. Calcium has been shown to affect exocytosis of glutamate (http://diabetes.diabetesjournals.org/content/50/5/1012.full) -so perhaps an alternate route is to serach for or design phosphate-gated calcium channels. Protein Kinase A has been shown to phosphorylate Calcium ion channels (http://www.ionchannels.org/showabstract.php?pmid=7621818). So a basic pathway leading to transcription of PKA instead of B-gal, combined with the proper type of Calcium Receptor, could lead to a pathway that exocytoses glutamate.

The ultimate goal of this is to develop a functioning mammalian cell that uses a Cph8 analog to exocytose glutamate in the absence of light. The route from bacterial to mammalian cells is not a direct one - [investigation of the differences between bacterial and mammalian cells in 2CS is needed]

Concerns
1. We need to figure out a way to get the responder protein into the nucleus of the mammalian cell.

2. PKA is already expressed at pretty high levels in many mammalian cells. Expressing more of it could be deleterious to the cells.

3. Tissue rejection of the modified cells could defeat the entire purpose of the therapy (if this gets that far).

4. It would be difficult to engineer a mammalian cell with all the necessary components for this system to work.

Further research is required to figure a way around these challenges.

Plan

 * 1) Harvest Cells from Patient
 * type?
 * 1) Transgenesis to induce expression of cph8 and Phosphorylation-gated Ca++ receptor.
 * how to get it into the Membrane?
 * 1) Eliminate Crosstalk.
 * 2) Induce G0
 * 3) Create Inorganic Box
 * 4) Reimplant