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Shyamtanu Datta, Bo Chen, Marian Renwick, Hui Peng, Rafael Ufret-Vincenty, John Hulleman; A destabilized domain-based approach to control protein abundance in aged mice and models of retinal degeneration. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3392. doi: https://doi.org/.
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Previously we applied a proof-of-concept conditional gene therapy approach that uses a destabilized domain version of E. coli dihydrofolate reductase (DHFR) fused to yellow fluorescent protein (YFP) to positively control protein abundance in young, healthy mouse eyes using the orally available small molecule antibiotic, trimethoprim (TMP). We next tested how well this DHFR-based system regulates protein abundance in the retina of aged mice and in environmental/genetic models of retinal degeneration.
A group of aged (9 and 18 mo) C57BL6/J mice were intravitreally injected with 7.6x10^9 viral genomes of rAAV2/2 [MAX] expressing DHFR-YFP and a concomitantly expressed mCherry. After >4 wk of transduction, mice were treated overnight with TMP (0.4 mg/mL) or normal drinking water. Mice were euthanized and their retina were harvested for western blotting. A separate group of similarly injected 8-10 wk old mice were designated for use in the severe fundus camera delivered, light induced retinal degeneration (FCD-LIRD) model. After >4 wk of transduction, FCD-LIRD-designated mice were injected with fluorescein (2 mg, i.p.) for 10 mins and subsequently exposed to 50,000 lux of light for 3 min. Degeneration was monitored over the next 7 d, after which mice were given TMP or normal drinking water overnight, imaged and sacrificed for western blotting.
In both cohorts of aged mice, DHFR-YFP was effectively turned-over in the absence of the stabilizing molecule, TMP. Protein levels of DHFR-YFP in non-TMP virus injected eyes were not significantly different than background signal observed in control HBSS-injected eyes. Overnight TMP significantly elevated DHFR-YFP protein levels by >30 fold vs. non-TMP DHFR-YFP levels (p<0.01, t-test). During FCD-LIRD, no DHFR-YFP was observed at any timepoint during degeneration (in the absence of TMP), yet overnight TMP administration stabilized DHFR-YFP after 7 d of degeneration.
This DHFR-based approach allows for efficient conditional control of protein abundance in aged mice and mouse models of retinal degeneration. These data lay the foundation for using this strategy for regulating gene therapies in a variety of mouse models of retinal degeneration.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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