Neuroprotective Gene Therapy by Overexpression of the Transcription Factor MAX in Rat Models of Glaucomatous Neurodegeneration

Abstract

Purpose Based on our preview evidence that reduced nuclear content of the transcription factor Myc-associated protein X (MAX) is an early event associated with degeneration of retinal ganglion cells (RGCs), in the present study, our purpose was to test whether the overexpression of human MAX had a neuroprotective effect against RGC injury. Methods Overexpression of either MAX or green fluorescent protein (GFP) in the retina was achieved by intravitreal injections of recombinant adenovirus-associated viruses (rAAVs). Lister Hooded rats were used in three models of RGC degeneration: (1) cultures of retinal explants for 30 hours ex vivo from the eyes of 14-day-old rats that had received intravitreal injections of rAAV2- MAX or the control vector rAAV2- GFP at birth; (2) an optic nerve crush model, in which 1-month-old rats received intravitreal injection of either rAAV2- MAX or rAAV2- GFP and, 4 weeks later, were operated on; and (3) an ocular hypertension (OHT) glaucoma model, in which 1-month-old rats received intravitreal injection of either rAAV2- MAX or rAAV2- GFP and, 4 weeks later, were subject to cauterization of the limbal plexus. Cell death was estimated by detection of pyknotic nuclei and TUNEL technique and correlated with MAX immunocontent in an ex vivo model of retinal explants. MAX expression was detected by quantitative RT-PCR. In the OHT model, survival of RGCs was quantified by retrograde labeling with DiI or immunostaining for BRN3a at 14 days after in vivo injury. Functional integrity of RGCs was analyzed through pattern electroretinography, and damage to the optic nerve was examined in semithin sections. Results In all three models of RGC insult, gene therapy by overexpression of MAX prevented RGC death. Also, ON degeneration and electrophysiologic deficits were prevented in the OHT model. Conclusions Our experiments offer proof of concept for a novel neuroprotective gene therapy for glaucomatous neurodegeneration based on overexpression of MAX.

Optic Nerve and Retinal Ganglion Cell Protection, Rejuvenation, and Regeneration as Glaucoma Treatment Strategies

“…RGCs and their axons in DBA/2 J mice and other mice with microbead-induced ocular hypertension (OHT) could be protected by genetic expression of the anti-apoptotic soluble Fas-ligand [44], by over-expression of the complement C3 inhibitor CR2-Crry [45], and by retinal expression of scAAV2-C3 (exoenzyme C3 transferase), which significantly reduced the number of apoptotic RGCs and decreased cell loss in the ganglion cell layer after ischemia/reperfusion injury [46]. Additional examples of gene therapies for RGC preservation in the face of death signals after GON induction in various animal models have involved delivering X-linked inhibitor of apoptosis (XIAP; potent caspase inhibitor) [47], transduction of RGCs with the protective transcription factors BCL2L1 [48], and Myc-associated protein X (MAX) [49] and over-expression of NMNAT1, the key enzyme in the NAD + biosynthetic pathway to enhance RGC rejuvenation through increased intrinsic energy production [50]. A most advanced form of gene therapy that proved successful in animals has been the co-delivery of the neurotrophin BDNF and its receptor to impart neuroprotection to RGCs [51].…”

Future directions of glaucoma treatment: emerging gene, neuroprotection, nanomedicine, stem cell, and vascular therapies

“…Other neuroprotective gene therapy strategies that have shown promise in preclinical studies include increased expression of antioxidant genes [31,32], anti-inflammatory genes [33–36,37 ▪ ], cell cycle regulators [38], and protease inhibitors [39 ▪▪ ]. Recently, Margeta et al demonstrated that targeted deletion of the Apoe gene or carrying the APOE4 allele protected against RGC loss despite elevated IOP in mouse glaucoma models [40 ▪▪ ].…”

From Bench to Bed: The Current Genome Editing Therapies for Glaucoma

“…In addition to neurotrophins, anti-axon retraction, anti-inflammation treatment, anti-apoptosis treatment, antioxidation treatment, and gene transfer of MAX, BRN3B, Hsp-70, PDEF, and EpoR76E also have the ability to protect RGC survival. Based on the finding that decreased content of the transcription factor Myc-associated protein X (MAX) is associated with degeneration of RGCs, a recent study demonstrated that overexpression of human MAX had a neuroprotective effect against RGC injury ( Lani-Louzada et al, 2022 ).…”