DISCUSSION

Hauswirth: In the rds gene replacement therapy, what level of rds expression are you achieving relative to wild-type levels in the animal model? It doesn’t look like it is very high.

Ali: That’s a good question. We are going to try to ¢nd out.

Hauswirth: This provides a potential explanation for what photoreceptors still die, because of a haploinsu⁄cient e¡ect.

Ali: It is possible, but the degeneration in the rds heterozygote is very slow. Hauswirth: But this has 50% of normal levels and from your data it didn’t look

like you had anywhere near this level.

Ali: These are not simple experiments to do, but we are thinking about doing a quantitative RT-PCR to measure this.

Bird: How does the ERG compare between the treated and the heterozygote? Ali: The heterozygote is clearly better.

Molday: Have you checked whether ROM1 expression is increased?

Ali: No. It would be di⁄cult to have intact outer segments without increased ROM1.

Molday: No, outer segments can form in the absence of ROM1.

McInnes: In the ROM1 knockouts the outer segments are pretty healthy looking. Travis: Would you say that in the injected area you are transducing close to all of

the photoreceptors, or just a subset?

Ali: We are not transducing all the photoreceptor cells in the area. It is variable.

Travis: Having a situation where there are untransduced photoreceptors around a few transduced receptors expressing at a low level will be especially di⁄cult to unravel.

Ali: It is a bit of an unusual situation because the rds mouse has no function. The key issue is whether we can improve the function and slow the degeneration. Just slowing the degeneration means very little in this context.

Travis: The fact that you are making outer segments is signi¢cant, though. Ali: We are making progress.

Kaleko: With respect to the slow loss of the improvement in the rds model, is it possible that the promoter shut o¡ over time or that there was an in£ammatory response to the foreign transgene product in these animals?

Ali: In terms of in£ammatory response, we don’t really know, although I wouldn’t absolutely rule it out, I think it is unlikely. We have carried out some experiments to show that we are not getting inactivation of the promoter. The in£ammatory response is unlikely because we can get long-term expression of green £uorescent protein (GFP), although I accept your point that perhaps peripherin is more immunogenic than GFP.

Kaleko: Sometimes in£ammatory responses can be fairly subtle. I have a second question. Do you think that the vector entry pathway into the photoreceptors is through the help of heparan sulfate glycosaminoglycans?

Hauswirth: For type 2 it de¢nitely is. Bok: Which isotype are you using? Ali: Type 2.

Kaleko: Will there be a problem with subretinal injection under the macula? Ali: I’m not the best person to answer this.

Sahel: Once you detach the macula the outer segments start to disappear. It may take many months before they regenerate. Did you detach the retina in your injections?

Ali: We do not cause a full detachment but we do detach between 40^60% and the ERG function seems to be normal.

Sahel: Certainly, the dog can elicit a lot of in£ammatory responses by itself depending on the point of entry of the injection. It is unreliable.

Ali: Sometimes complete retinal detachments are performed when macular translocations are performed.

Sahel: I don’t advise this.

Bird: I don’t think the surgeon would see it as a problem. They are an adventurous group. But it is the case that surgical detachment of the macula with reattachment does not appear to be a very noxious exercise, as opposed to natural detachment where the subretinal space is open for a long time. If this is seen as a way of preserving function that would inevitably be lost, this will not be seen as a major problem.

Sahel: Steve Fisher (Rex et al 2002) has shown that once the retina is detached that there many markers of the in£ammatory response present.

Ali: He keeps the retina arti¢cially detached. We are carrying out a transitory detachment. It re-attaches within 24 hours. This type of detachment may not be as big a problem as one might ¢rst think. We have not found much evidence for damage.

Sieving: That is hedging the question. Why not do the experiment and publish the results? It is an important issue. It is hard for me to imagine that one can elevate the photoreceptors out of the nice RPE pockets that ensheathe the rod outer segments and then expect that the rods will slip right back into the sheathes afterwards. Might not the pigment epithelium sheathes collapse and then later on regrow around the outer segments? About 20 years ago I recorded an ERP (early receptor potential), in a patient several years after he underwent macula reattachment surgery. The ERP provides a measure of rhodopsin quantity in the rods and thereby gives information about rod outer segment length. Although this patient had 20:20 acuity, the ERP amplitude was only half normal, indicating that the macular photoreceptor outer segments were only half their normal length. In

other words, the e¡ect of his detachment persisted for many months. It is worth getting some data on this issue of structural retinal e¡ects following arti¢cial detachment and reattachment.

Sahel: There are two indirect ways to study this. First, in patients with retinal detachment, even if the macula is detached just for one day, it can take weeks or months to see total recovery. Second, if you look at the CTF to VCTF reattachment there is sometimes a tiny amount of liquid under the macula. Clinically it looks OK, but if you look carefully it is not that good.

Ali: It is obvious that if you have a normal healthy retina, the last thing we want to do is to detach it. If you have a severe disease process and there is no other way of doing anything then one might contemplate a retinal detachment.

Dryja: We just have to do the studies. One thing that surgeons might agree with is that repeated macular injections would not be good. It hard to imagine that one could do this every six months and expect the retina to last a lifetime.

Zack: Given the results that you have found, that a subretinal injection causes ganglion cell expression in the dog, how good is photoreceptor expression if you inject intravitreally? This might work for the macula and fovea in particular, since this is so thin.

Ali: That result is just because we are putting so much in and we are getting leakage out into the vitreous.

Zack: Have you tried injecting this much virus into the vitreous in the dog?

Ali: No.

Hauswirth: We have done the experiments in pigs and we only get ganglion cell transduction. The idea of doing it in the macula has some merit. Since there are many fewer cells that have to be passed through from the macula to get into photoreceptors, perhaps if you added a little heparin to compete with the initial binding at the proximal surface you could get penetration of a small viral vector.

Dryja: If it works, the retinal surgeons will ¢gure out ways to administer the virus.

Aguirre: There seemed to be a great variability in the amount of rhodopsin and the outer segment length in the rds mouse that was treated. Is this a factor of time after injection?

Ali: Yes, you are right.

LaVail: In your Mertk experiments, it didn’t look like there were many inner and outer segments there. From our experience in RCS rats, you are not going to get nearly the distribution that you get in any normal retina or other degenerate retinas because of all the debris in RCS rats. If this is the case, I was amazed at your ERG ¢ndings. You got demonstrable ERG b-waves with AAV RPE65.

Ali: But we had better results with the CMV.

LaVail: To have a relatively small rescue in a restricted area, and to have that much of an ERG response seems incompatible.

Ali: The results I showed were from repeated double injections, so we did four in all. To try to optimize our rescue we started to carry out multiple injections.

Aguirre: Are these at the same site? Ali: No, di¡erent sites.

Reference

Rex TS, Fariss RN, Lewis GP, Linberg KA, Sokal I, Fisher SK 2002 A survey of molecular expression by photoreceptors after experimental retinal detachment. Invest Ophthalmol Vis Sci 43:1234^1247