Table 1.

Summary of in vivo and in vitro studies utilizing an adeno-associated viral (AAV) vector encoding brain-derived neurotrophic factor (BDNF) as gene therapy.

Reference/Study	Country	Injury Model and Animal	Groups and Sample Size	Parameters for Efficacy	Main Result	Remarks
1. Viral vector encoding brain-derived neurotrophic factor (BDNF)
a. Adeno-associated viral vector gene therapy (AAV2) encoding BDNF (AAV2-BDNF) Wójcik-Gryciuk A. et al., 2020 [11]	Poland	Microbead induced elevated IOP, rat	12 negative control, 14 damaged only, and 13 damage + treatment	RGC count BDNF and TrkB protein levels	Significant attenuation of RGC loss in AAV2-BDNF treated rats (1267 ± 60 RGCs/mm2) vs. glaucoma induced rats (758 ± 124 RGCs/mm2). Unilateral injection of BDNF, bilaterally upregulated BDNF levels and restoration of TrkB levels back to normal levels.	Neuroprotection of RGCs indicated by the attenuated loss of RGCs. Unilateral injection of BDNF was able to upregulate BDNF in both injected and non-injected eyes.
b. AAV2 gene therapy encoding BDNF and tropomyosin related receptor kinase-B (AAV2 TrkB-2A-mBDNF) Osborne A. et al., 2018 [12]	United Kingdom	In vitro oxidative damage via hydrogen peroxide, immature SH-SY5Y human neuroblastoma cells	4 negative control, 4 empty vector control, 4 TrkB + BDNF treated, 4 TrkB treated only, 4 BDNF treated only	Neuroprotective effect via TUNEL measurement	TrkB + BDNF treated SH-SY5Y cells significantly reduced TUNEL positive apoptotic cells.	The neuroprotective effects of just BDNF or TrkB treatment alone were not assessed when damaged with hydrogen peroxide.
c. AAV2 gene therapy encoding BDNF and TrkB (AAV2 TrkB-2A-mBDNF) Osborne A. et al., 2018 [13]	United Kingdom	Optic nerve crush, mice and rats	Cohort 1: 16 positive control Cohort 2: 12 TrkB + BDNF treated mice Cohort 3: 36 TrkB + BDNF treated mice Cohort 4: 25 BDNF treated mice Cohort 5: 22 TrkB + BDNF treated mice study mice Cohort 6: 15 TrkB + BDNF treated mice and 15 BDNF treated only Cohort 7: 20 TrkB + BDNF treated mice and 20 TrkB treated only Cohort 8: 30 TrkB + BDNF treated mice	Brn3a positive RGC count Glial fibrillary acidic protein (GFAP) activation Visual function via electroretinography (ERG) Axon and RGC counts in rats with laser-induced ocular hypertension (OHT)	BDNF treatment significantly rescued Brn3a+ RGCs. TrkB + BDNF treatment significantly rescued Brn3a+ RGCs and there was a significantly greater rescue effect with both TrkB + BDNF compared to BDNF treatment alone. No significant difference in GFAP levels in TrkB + BDNF groups compared to GFP control group and negative control up to 24 weeks. Significant improvement in positive scotopic threshold response (pSTR) in TrkB + BDNF treated mice in optic nerve crush compared to just TrkB alone. No significant changes in negative A- or positive B-wave responses in any of the treatment groups. TrkB and BDNF were able to significantly protect and restore axon counts and RGCs in rats.	TrkB + BDNF were able to significantly rescue more RGCs than BDNF treatment only No adverse effects on retinal health with TrkB + BDNF treatment as suggested by a lack of change in GFAP levels. Only TrkB + BDNF and TrkB treatment were compared with BDNF treatment only. Only TrkB + BDNF and TrkB treatment only were compared with BDNF treatment only.
d. Tyrosine triple mutated AAV2-BDNF (tm-scAAV2-BDNF) gene therapy Shiozawa A.L. et al., 2020 [14]	Japan	N-methyl-D-aspartate (NMDA) induced retinal injury, mouse	6 negative control, 6 damage, 9 damaged + treatment	Inner retinal layer thickness Brn3a positive RGC count Visual function via scotopic ERG GFAP activation	Significant attenuation of inner retinal thickness loss in damage + treatment mice vs. damage only mice. Significant prevention of Brn3a+ RGC loss in damage + treatment mice vs damage only mice. Significantly higher A- and B-wave amplitudes in the damage + treatment mice vs. damage only mice. Significant reduction in GFAP in the damage + treatment mice vs. damage only mice.	Protection and prevention of histological changes caused by NMDA. Neuroprotection of RGCs indicated by an attenuated loss of RGCs. Suggested preservation of visual function. Reduction in retinal damage using GFAP as a maker for retinal stress.
e. tm-scAAV2-BDNF gene therapy Igarashi T. et al., 2016 [15]	Japan	Increased IOP via saline infusion to induce retinal ischemia/reperfusion (I/R), mouse	6 control, 6 damage, 6 damage + empty vector, 6 damage + treatment	Inner retinal layer thickness Brn3a positive RGC count Visual function via scotopic ERG GFAP activation	Significant attenuation of inner retinal thickness loss in damage + treatment mice (45.4 ± 4.2 µm) vs. damage + empty vector mice (30.2 ± 3.0 µm). Significant prevention of Brn3a+ RGC loss in damage + treatment mice (22.6 ± 0.5 cells) vs. damage + empty vector mice (11.2 ± 0.8 cells). Significantly higher B-wave amplitudes in damage + treatment mice (862.6 ± 146.6 µv) vs. damage + empty vector mice (484.8 ± 201.6 µv). Significant reduction in GFAP in damage + treatment mice (4.8 ± 4.4) vs. damage + empty vector mice (12.3 ± 0.6)	Protective effects of BDNF indicated by the thicker retinal structure and no significant difference with normal mice. Neuroprotection of RGCs indicated by an attenuated loss of RGCs. Suggested preservation of visual function. Reduction in retinal damage using GFAP as a maker for retinal stress.
f. AAV2-BDNF gene therapy and/or an AAV2 vector encoding a mutated phosphor-resistant version of collapsing response mediator protein 2 (AAV2-CRMP2T555A) Chiha W. et al., 2020 [16]	Australia	Unilateral partial transection of the optic nerve, rats	11 negative control, 11 control vector with GFP, 6 BDNF + GFP, 9 BDNF only, 9 CRMP2T555A only, 10 BDNF + CRMP2T555A	Visual function via optokinetic nystagmus response RGC count Axonal density in the optic nerve Structural disruptions at the node of Ranvier complex Myelin thickness via G-ratio Oxidative stress and inflammatory cell markers	Injured rats with BDNF (BDNF only and BDNF + CRMPT2T555A) had a significantly increased total number of pursuits compared to injured rats with control vector with GFP injections. In the central retina, BDNF, only, CRMPT2T555A only, and BDNF + CRMPT2T555A significantly increased RGC counts compared to injured control vector groups in the central retina when counting βIII-tubulin+ RGCs. When counting Brn3a+ RGCs, only CRMP2T555A only and BDNF + CRMP2T555A significantly increased RGC counts. In the ventral retina, BDNF only significantly increased RGC counts with either βIII-tubulin+ or Brn3a+ RGCs. BDNF only, CRMP2T555A only, and BNDF + CRMP2T555A treatment significantly restored the number of optic nerve axons. BNDF + CRMP2T555A treatment significantly restored the paranodal gap when defined as two Caspr+ paranodes. BDNF only and BNDF + CRMP2T555A treatment significantly restored the paranodal gap when defined as βIII-tubulin+ axons flanked by Caspr+ paranodes. BDNF only, CRMP2T555A only, and BNDF + CRMP2T555A treatment significantly reduced the G-ratio and myelin thickness. Only BNDF + CRMP2T555A treatment was able to significantly reduce HNE, an oxidative stress marker, while there was no affect from any treatment groups on the microglia marker, Iba1.	Partial ON transection has been previously reported to reduce the optokinetic nystagmus response. Total pursuits included smooth, partial, and micro pursuits. Smooth and fast rests were not significantly different. βIII-tubulin+ RGCs were also counted as not all RGC subtypes express Brn3a, and RGC loss after injury is often preceded by a downregulation in Brn3a. For axonal density, βIII-tubulin+ RGCs were also counted as not all RGC subtypes express Brn3a, and RGC loss after injury is often preceded by a downregulation in Brn3a. Paranodal gaps defined as βIII-tubulin+ axons flanked by Caspr+ paranodes represents a more rigorous definition for the Node of Ranvier. G-ratio is the ratio of the inner axonal diameter to the total diameter and it is used to assess axonal myelination. HNE increases in ONs vulnerable to secondary degeneration.