Purchase this article with an account.
M. S. Gorbatyuk, T. J. Knox, J. Thomas, Jr., V. Chiodo, W. W. Hauswirth, A. S. Lewin; Protection of Phortoreceptors in P23H Transgenic Rats by Over-Expression of Heat Shock Proteins Delivered by Adeno-Associated Virus. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5352. doi: https://doi.org/.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
A substantial fraction of mutations in the gene for rhodopsin leading ADRP are though to lead to misfolding of the opsin protein. The P23H mutation, which is relatively common in North America is such a mutation. If such misfolded proteins exceed the clearance capacity of the ubiquitin proteosome system, then they may activate the Unfolded Protein Response (UPR) and lead to apoptotic cell death. One method to overcome this pathogenic process is to promote the correct folding of mutant opsin proteins. The goal of this project is to develop a gene therapy based of the delivery of Heat Shock Proteins (Hsp) such as Hsp70 and Grp78 that would facilitate the folding mutated opsin protein and lead to the protection of the photoreceptors from ER stress.
cDNAs for human Hsp70 and Grp78 genes were cloned into AAV plasmids under control of the hybrid CMV-beta actin(CBA) promoter. Plasmids were packaged in AAV serotype 5 capsids. Line 3 P23H transgenic rat pups were injected in one eye at postnatal day 15. Animals were monitored up to three months. Full field dark-adapted electroretinography (ERG) was used to detect changes in the response to light in the treated eye compared to the control eyes which were injected with a control virus. Then rats were sacrificed and eyes were enucleated for histological and immunostaining analysis. Total protein and RNA extracts were also taken to study the changes in the protein and mRNA levels in the treated photoreceptors. These extracts were also used to monitor markers of the UPR .
AAV5-Grp78 and AAV5-Hsp70 injections caused measurable over-expression of Hsp70 and Grp78 in the retina. The results of ERG analysis showed an elevation of a- and b-waves amplitudes in HSP treated eyes relative to control eyes. However, the rescue effects obtained from two injections were not similar. A- and b-wave amplitude was increased by 20 to 40% in case of AAV-HSP70 but by 43-115% following AAV-Grp78 injection. Morphometic analysis of the Outer Nuclear Layer (ONL) across the retina indicated that these therapeutic effects were due an increase of up to 45% in the ONL thickness. The ER stress protein CHOP was reduced in the protein extracts of eyes treated with GRP78.
The over-expression of Hsp70 and Grp78 in photoreceptors appears to reduce retinal degeneration associated with P23H rhodopsin. This supports our hypothesis that these cells are undergoing ER stress caused by misfol ded opsin. Optimization of the gene delivery might be necessarily to elevate the therapeutic effect.
This PDF is available to Subscribers Only