Hi Dr. Leveillard,

I wanted to get your detailed feedback regarding your recent interview for this article.  I am concerned that both of our interviews were edited in such a way that they lost most of their original technical content.


To provide an executive summary of what we are doing:
EYS is a photoreceptor-specific growth factor that promotes outer segment regeneration.  This proteoglycan is homologous to the proteins perlecan and agrin, which are responsible for triggering tissue repair and wound healing via the notch signalling pathway.  EYS has isoforms that are bound to the extracellular matrix, similar perlecan and agrin, as well as smaller isoforms that are secreted continuously during normal OS disc renewal.  The smaller isoforms, numbered 2 and 4, are approximately the same molecular weight as ranibizumab (~65 kDA vs. ~50 kDa), so EYS should be able to penetrate the retina when administered by the intravitreal route.

RP25 results from homozygous loss of function in EYS, and this is our initial therapeutic target.  We will treat RP25 by intravitreal protein replacement therapy, which is considerably easier to develop, as well as obtain regulatory approval for, than gene or cell therapies.  Additionally, since EYS is a photoreceptor-specific growth factor, it may have applications in treating other retinal diseases as well, in addition to RP25.

Initially we will be focused on very small-scale proof of concept experiments in order to demonstrate the feasibility of EYS replacement.  If these initial experiments show promise, we will use them to obtain venture capital investment to fund proper preclinical development, with better facilities and equipment.  Our proof of concept experiments will use zebrafish RP25 disease models, as well as pre-synthesized EYS protein ordered online from Genscript.

We are planning to start by studying the pharmokinetics of GFP-tagged EYS in healthy zebrafish using retinal cryosections.  If the fluorescent tagged EYS is able to migrate from the vitreal injection site, to localize in the outer segment as we intend, then we will begin to evaluate its therapeutic capabilities.

For our second experiment, we will use an off the shelf CRISPR/Cas9 knockout kit to disable EYS in zebrafish embryos.   We are going to compare the rates of visual decline in treated fish versus sham injection controls, based on their electroretinogram amplitudes.  EYS loss of function results in generalized cone-rod dysfunction, so we will record their ERG responses to a photopic white flash.  Our goal is to demonstrate a statistically significant difference in the rate of visual decline, which will warrant further investigation during the preclinical phase.

Only once we have demonstrated statistical significance and obtained proper funding/facilities, will we attempt to purify our own EYS from cell cultures.  Since EYS is a proteoglycan, it is very important that we obtain proper glycosylation from our expression system: this is why we are planning to use HEK293 cultures in the future.  Additionally, at no point in any of this are we proposing to perform unregulated experiments on humans.  We are only concerned with animal models, and we are planning to follow established best practices for humane treatment of our zebrafish.


I understand your skepticism, and quite frankly, if I were in your position I wouldn't believe this story either.  However, with that being said, nobody else is researching RP25 so I am taking matters into my own hands.  We are seeking technical criticisms from experts so that we can address the issues in our plan, and we would be very grateful if you could offer us your detailed feedback.  Would you have time to discuss this project further?


Thanks,