Table 3.

Summary of in vivo studies utilizing an AAV vector encoding nuclear factor erythroid 2-related factor 2 (NRF2) as gene therapy.

Reference/Study	Country	Injury Model and Animal	Groups and Sample Size	Parameters for Efficacy	Main Result	Remarks
3. Viral vector encoding NRF2
a. AAV2-NRF2 and AAV2-Sirtuin 1 (SIRT1) gene therapy McDougald DS. et al., 2018 [19]	United States	Induction of experimental autoimmune encephalomyelitis (EAE), mice	10 negative control, 10 injury + vehicle, 10 GFP only, 10 injury + GFP, 25 AAV2-NRF2, 25 AAV2-SIRT1	Visual acuity RGC count via Brn3a Immune cell infiltration via H&E staining and demyelination measured via LFB staining	Injured mice treated with AAV2-SIRT1 had significant improvement in optokinetic responses (OKR) compared to injury + GFP mice. Injured mice treated with AAV2-NRF2 had significantly higher numbers of Brn3a+ RGCs compared to injury + vehicle. AAV2-SIRT1 treatment did not significantly increase RGCs compared to injury + vehicle mice. Both NRF2 and SIRT1 had no significant impact or attenuation of immune cell infiltration or demyelination of ON sections.	There was no significant improvement in OKR with AAV2-NRF2 treatment. There was no significant difference with NRF2 treatment compared to injury + GFP, despite any purposeful therapeutic gene encoded in the AAV2-GFP vector. There was no significant difference among all groups that were injured, indicating no protective or therapeutic response of NRF2 or SIRT1 treatment.
b. AAV2-pMcp-NRF2 gene therapy Fujita K. et al., 2017 [20]	Japan	ON crush model, mice	8 negative control, 8 pMcp promoter treatment, 8 CMV promoter treatment	mRNA expression of target gene (NRF2) RGC death measured via Sytox Orange RGC count measured via Brn3a, Brn3b, and Thy1 Mcp1 promoter and CMV promoter driven AAV2-NRF2 treatment Gene expression differences in Mcp1 and CMV promoter driven AAV2-NRF2 treatment	Mice with ON crush had significantly higher expression of NRF2 compared to mice treated with AAV2-pMcp-NRF2 without injury. Treated mice had significantly reduced numbers of Sytox positive cells in ON crush eyes. RGC makers (Brn3a, Brn3b, and Thy1) were significantly increased with NRF2 treatment compared to non-injured controls. Treatment with Mcp1 promoter or CMV promoter driven AAV2-NRF2 conferred no differences in mRNA expression of RGC markers or visual acuity and contrast sensitivity. CMV promoter driven AAV2-NRF2 significantly increased Ho-1, Atf4, and p53 compared to control and Mcp-1 promoter driven AAV2-NRF2.	An increase in NRF2 gene expression selectively in injured eyes suggests positive selectivity with the pMcp promoter. Sytox Orange is a nucleic acid stain for dead cells. Relative mRNA levels were presented, not RGC counts. Despite a Mcp1 promoter being utilized to be more specific for stressed RGCs, the effects seem to be no different from a CMV promoter. Ho-1 is a direct transcription target of NRF2, Aft4 is an ER stress marker, and p53 is involved in the cell death pathway.