Hi again!

I need to make sure I understand what's going on.

You have retinitis pigmentosa type 25. That disease is caused by a mutation in the EYS gene, which is supposed to produce a protein named EYS too. Without this protein, which is supposed to protect the retina from damage, your photoreceptor cells are dying, and that's very bad. Your plan is to produce EYS artificially with kidney cells, and to find a way to deliver it to the retinal outer segment.

The part about producing EYS is bothering me the most: are you going to to synthesize the protein? To glycosylate EYS protein you bought to obtain a new form of EYS? Is there any simple way to explain how it's going to go inside kidney cells? Are you still going to use CRISPR-Cas9? I am very sorry for bothering you, you must be really busy.

Thank you very much,

Hi, also: where are you going to perform your experiments?


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Good questions!

EYS has a few different jobs, one of which is protecting certain types of nervous tissue from osmotic forces.  When there is a difference in the concentration of dissolved salts inside the cell versus outside the cell, water will rush either into or out of the cell, either inflating it or crushing it.  EYS selectively binds to the outer segment of the retina where it forms a protective cell wall that reinforces against osmotic pressure, making these cells stiffer and more rigid.

We also think it has an additional role in photoreceptor maintenance and renewal.  In healthy retinas, the part of the photoreceptor cell that captures light and allows you to see gets replaced over time.  This is because the phototransduction machinery wears out as you use it.  In some types of retinitis pigmentosa, the photoreceptors aren't able to as effectively replace their worn out components, and so they degrade with time.  EYS might have a role in stimulating this regeneration process.

So what we are going to do is genetically modify some cell cultures so that they produce EYS, grow the cultures in a vat, purify the protein from the cultures, and then inject the protein into the eye.  People with RP25 have mutations in the EYS gene that prevent them from producing the protein.  By growing the protein for them and injecting it, we can hopefully treat their disease.  We are able to do this because EYS is naturally secreted outside the cell, as opposed to doing some job inside the cell like other retinitis pigmentosa causing mutations.



Human embryonic kidney cells are used by a lot of pharmaceutical companies to produce things like biologic drugs and some types of vaccines.  They were taken from a patient in 1973 and grown outside the body since then.

We are going to use CRISPR/Cas9 in two places, but not directly in treatment.

First, we are going to use CRISPR to insert our EYS expression vector into the cell cultures.  That will be a snippet of DNA containing:

    a promoter sequence to enhance protein synthesis
    EYS cDNA (basically a compressed copy of the gene with the unnessescary parts removed)
    a tag sequence that will allow us to identify and purify the protein later on
    a sequence coding for resistance to methotrexate

To actually modify the cells, we place the kidney cells, Cas9 enzymes, and expression vector DNA together in solution.  We then put the mixture in an electroporation machine which subjects the cells to intense electric fields, opening up little gaps in their cell membranes that allow the genes and Cas9 to seep through.  Cas9 then does all the work of modifying the DNA in the cells, and then finally we add methotrexate to the solution to kill off all the cells that didn't incorporate our EYS DNA.
Second, we will also use CRISPR/Cas9 on zebrafish embryos to deactivate their EYS gene and produce models of human RP25 that we can study.



This project is going to have three main phases:

    proof of concept
    preclinical trials
    clinical trials and scaling up

Right now we are in the proof of concept phase.  During this phase our main goal is to prove that this treatment is possible, and to do so as simply and cheaply as we can manage.  Once we can show some basic evidence of EYS altering the course of RP25 retinal degeneration in disease models, we will be able to seek funding for better facilities and equipment to do more advanced studies.
As long as we are still in the proof of concept phase, we will be using very makeshift equipment and facilities.  I'm in the process of setting up a work area in my garage.  We don't need large amounts of the protein for the first two experiments, so the plan is to order it pre-synthesized from an online supplier.  Not only will that save us time, but it will also save us a lot of money on lab equipment while we are starting out.  We estimate that will allow us to complete the proof of concept phase for under $15k USD, which we plan to raise by crowdfunding.

Also, one of the benefits of being a mechanical engineer is that I can make some of the equipment by myself very cheaply.  I'm working on designs for a simple electroretinogram, as well as a subretinal injection device if we end up needing it.