Spatially and Temporally Regulated NRF2 Gene Therapy Using Mcp-1 Promoter in Retinal Ganglion Cell Injury

Abstract

Retinal ganglion cell degeneration triggered by axonal injury is believed to underlie many ocular diseases, including glaucoma and optic neuritis. In these diseases, retinal ganglion cells are affected unevenly, both spatially and temporally, such that healthy and unhealthy cells coexist in different patterns at different time points. Herein, we describe a temporally and spatially regulated adeno-associated virus gene therapy aiming to reduce undesired off-target effects on healthy retinal neurons. The Mcp-1 promoter previously shown to be activated in stressed retinal ganglion cells following murine optic nerve injury was combined with the neuroprotective intracellular transcription factor Nrf2. In this model, Mcp-1 promoter-driven NRF2 expression targeting only stressed retinal ganglion cells showed efficacy equivalent to non-selective cytomegalovirus promoter-driven therapy for preventing cell death. However, cytomegalovirus promoter-mediated NRF2 transcription induced cellular stress responses and death of Brn3A-positive uninjured retinal ganglion cells. Such undesired effects were reduced substantially by adopting the Mcp-1 promoter. Combining a stress-responsive promoter and intracellular therapeutic gene is a versatile approach for specifically targeting cells at risk of degeneration. This strategy may be applicable to numerous chronic ocular and non-ocular conditions.

Single AAV-mediated scarless genome editing in dysfunctional retinal neurons mediates robust visual restoration in mice

“…They were each sub-cloned into a pAAV-MCS Promoterless Expression Vector (Cell Biolabs Inc., San Diego, CA) containing an enhanced green fluorescent protein (EGFP) gene as a reporter, as previously described. 24, 27 AAV2/8 containing the reporter constructs were generated and purified following the method described below. Each virus was injected into 2 eyes of C57BL/6J mice.…”

“…AAV particles was extracted in PBS and purified with an AKTA prime plus chromatography system (GE Healthcare, Chicago, IL) on an AVB Separose HP column (GE Healthcare), as previously described. 24, 27…”

“…RT-PCR was carried out as described previously. 27 Total RNA was purified from the mouse retinas using the miRNeasy plus mini kit (Qiagen) and reverse-transcribed with SuperScript III (Thermo Fisher Scientific). qRT-PCR was performed with an initial denaturation step at 95° C for 20 s, followed by 40 cycles at 95° C for 3 s and 60° C for 20 s (7500 Fast Real-Time PCR System; Thermo Fisher Scientific).…”

“…Immunohistochemistry was performed as described previously. 27 Six weeks (1.5M) after the AAV injection, the eyes were fixed in 4% paraformaldehyde. They were then either embedded in OCT compound and sectioned using a cryostat to generate retinal sections, or the RPE/choroid complex was separated from the retina and then flattened by creating 4 incisions from the periphery to the optic nerve, thereby resulting in a clover-shaped RPE/choroid flatmount.…”

A drug-tunable gene therapy for broad-spectrum protection against retinal degeneration

“…However, it is a daunting task to design therapies for every individual gene or mutation that causes retinal dysfunction. To circumvent this challenge, additional strategies are based on gene/mutation-independent approaches, including expression of transcriptional regulators, inhibitors of apoptosis, regulators of oxidative defense, inhibitors of inflammation, and expression of neurotrophic factors (16)(17)(18)(19)(20)(21)(22)(23)(24).…”