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John D Ash, Casey Keuthan, Aisha Imam, Sanford L Boye, Shannon Elizabeth Boye, Clayton Santiago; Development of a drug-regulated, broad-spectrum neuroprotective gene therapy for retinal degeneration. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3961.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal degenerations are a large cluster of diseases characterized by the irreversible loss of retinal neurons. Neurotrophic factors, which elicit broad-spectrum protection against a variety of insults, have been proposed as potential therapies. While this has had success, the inability to control therapy expression has been a major limitation. To address this problem, we developed a drug-regulated neuroprotective factor named Retinal Protective Factor 2 (RPF2).
The RPF2 transgene consists of a synthetic neurotrophic factor fused to the E. coli DHFR destabilization domain and cloned into AAV compatible vectors with a chicken beta actin promoter. For in vitro studies, plasmids were transfected into rMC-1 cells and treated with varying concentrations of TMP to induce RPF2 stability. Levels of secreted RPF2 were detected by ELISA. Immunoblots from conditioned media experiments were used to measure STAT3 activation. Modified AAV2 or AAV6 capsids were used to deliver RPF2 into Balb/c or rd10 mouse models by intravitreal injections. TMP was provided to the animals in food or through intraperitoneal injection at varying doses. Electroretinography (ERG), OptoMotry (OKT), optical coherence tomography (OCT) and immunohistochemistry (IHC) were utilized to determine retinal function and morphology. For light damage (LD), animals were exposed to 1000 lux of light for 4 hours.
RPF2 expression was induced in a TMP dose-dependent manner and was reversible with drug withdrawal. Induced RFP2 secretion was able to activate STAT3 in un-induced cells. In vivo data show that induced RPF2 gene therapy was safe and did not induce inflammation. ERG, OCT, and OKT data also show that induced-RPF2 protected photoreceptors from light damage and prevented retinal degeneration caused by the PDE6brd10 mutation. Mice with induced RPF2 expression maintained visual acuity as measured by OKT, and have preservation of cones as seen by IHC.
We show that RPF2 is a potent neuroprotective factor for photoreceptors against multiple models of degeneration. Its expression is tunable by drug concentration and is reversible with drug withdrawal. This therapy’s broad-spectrum activity could function as a primary, standalone therapy, or used as an adjunct to a gene replacement or stem cell therapies.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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