Every vector/carrier has advantages and disadvantages. For the eye, AAV is currently the vector of choice. We designed our promoters accordingly. We did, however, use lentiviral vectors in early experiments (where we could not yet keep explants in culture for long), not because of the packaging size, but because of the fast onset of transgene expression.

Yes, we identified promising endogenous promoters to use as templates for promoter design and chose three additional promoters to test, based on the TNNT1, TRNP1 and TRPM1 promoters. None produced the desired ON-bipolar cell-specific expression.

No. We were specifically interested in expression in the retina.

We always treated both eyes.

If I am not mistaken, the promoter used in Macés paper is the original promoter designed by Kim et al. 2008. It is constructed of the SV40 basal promoter and the 200 bp murine enhancer. We only made experiments with its “upgraded” version, the 4xGRM6-SV40 promoter with the quadruple tandem repeat of the 200 bp enhancer (Cronin et al. 2014).

Up to approx. 1 year in wild type mice. We did not quantify changes in transgene expression levels, however.

Yes they could. In the experiments for the publication, we kept the explants in culture for 7 days. We improved the culturing however and could have cultured them for extended periods of time (multiple weeks).

No, not for this publication. The Kleinlogel lab is however establishing eclectrophysiological experiments for AAV-treated human retinal explants, and are doing patch clamp, MEA and V1-recording experiments for treated mice in the meantime.

No. We could not since we did bilateral injections.

No, but we also did not assess toxicity. However, I expect the toxicity from CMV to be mostly due to its strong (excessive?) expression strength, its unspecific expression and/or its viral origin. None of those are factors concerning our promoter.

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No, not at that time.

No, I did not. Since the human genome-based promoter is effective in both the human and the mouse retina, I would expect it to be effective in non-human primates retina as well.

Yes. We however needed to improve the longevity of our explant cultures first. The Kleinlogel lab have done or are planning those experiments now.

I did not and I think we will not. The enhancer is the larger part of the promoter so tandem repeats will eat up space fast and we showed very promising results in our rd1 mice. This indicates that expression levels are adequate. Too strong expression can lead to a burden for the cells and increased off-target expression.

We injected intravitreally. In general, we saw a somewhat higher transduction efficacy in non-central regions. We did not see noticeable differences in cell-type specificity in different regions.

The brightness of the screens was 10¹³ photons cm^-2 s^-1 and the contrast was set to maximum.

No.

As evidenced by our experiments using OPN1mw in rd1 mice as well as by other researchers using rhodopsin or channelrhodopsin, it appears that the degenerated retina easily provides/regenerates enough 11-cis-retinal for the optogenetic tools to be functional.

Vertebrate Opsin is a G-protein coupled receptor which, in photoreceptors, activates transducin. It has been shown as early as 2005 that ectopically expressed opsins can couple to and activate Gi/o pathways in other neurons as well. In ON bipolar cells, opsin can activate the natural intracellular G-protein coupled signalling cascade, taking advantage of the internal mechanisms of signal amplification and adaptation.

Yes, that is how we measured the visual acuity.

In human retinal explants by placing the virus directly onto the explant (which on day 0 of culturing was not submerged in medium). In mice by intravitreal injection.