Matthew Loper: Mod 3 project proposal
Project Details and Methods:
When siRNA is introduced to the body, most is filtered by the kidney or broken down by nucleases. Even if the siRNA manages to reach its target, the rate limiting step is getting it into a cell. We propose a redesigned M13 phage as the vector for delivering dsRNAs into cells. The p8 protein of modified M13 will hold onto the dsRNA, thus preventing nucleases from breaking it down. The p3 protein of this M13 will have a specific ligand to target a receptor on the cell membrane and allow endocytosis to occur. Thus, we hope to solve both problems in therapeutic siRNA delivery with our redesigned M13.
We will utilize a histidine switch to allow the dsRNA to be released from p8 once inside a cell. This works because histidine is neutral outside the cell at neutral pH (for example in the blood). Inside the cell, at pH ~5, histidine becomes protonated and realeases what it is bound to (dsRNA in this case).
The mechanics of how we will perform our modification have not been fully worked out. We will most likely use Phage Display to create a phage library and select for the M13 that has p3 that binds to our desired ligand (something like cholesterol to target the LDL receptor. Once we have found M13 with suitable p3 we will create another phage library and select for a M13 with p8 that attaches our histidine switch and siRNA. It is possible that we will discover that it is easier to use Synthetic Biology techniques to create our modified M13. Since the individual genes of M13 are known, we could design inserts to modify p3 and p8 at a genome level.
This project is extremely high risk and high reward. Using siRNA for therapeutic purposes has tremendous potential to change the way diseases are treated, however the biggest issue is the delivery of the siRNA to the target cells. Many different approaches have been tried (see http://www.sciencemag.org/products/lst_20071102.dtl) but since the discovery of RNAi occured recently, no one fully understands all the mechanisms and intricacies of the pathway. There is currently a race by numerous companies and groups to develop a method of siRNA delivery. The winner of this race will find significant financial and scientific rewards. If everything goes according to plan and we are able to develop a modified M13, we could win this race. The elegant aspect of our design is that our vector is a phage that can be infected into bacteria and mass produced unlike many other design tactics that involve complex methods to create the siRNA delivery vector.
However, if we are not able to develop a working M13 or find some unexpected flaw in our design, we will be back at square one. All of the time, effort and resources put into this project will have been lost.
Since we have not completely determined our modification method, we cannot exactly pinpoint the resources we will neeed. We will definitely need all the resources required to perform a phage display multiple times. However, if we determine phage display is not a suitable method for our modification we will need to use DNA synthesis to create inserts that can be used to modify M13. Regardless of the route we take, we will need sufficient lab space and funding for lab techs and research assistants.