Abstract
Purpose :
QTA020V is a novel gene construct designed to protect retinal ganglion cells (RGC) by long term activation of the mature brain-derived neurotrophic factor (mBDNF)/TrkB pathway. Our hypothesis is that upregulation of both neurotrophin and receptor will reduce RGC loss associated with glaucoma. We tested the ability of QTA020V to increase TrkB levels in RGC, to secrete mBDNF and to activate downstream signalling following intravitreal injection.
Methods :
Adeno-associated viral serotype-2 (rAAV2) vectors containing novel plasmids were injected intravitreally into mice (1-2μL, 1x10e10 vp/eye). Vectors were QTA020V (rAAV2-CAG-TrkB-2A-mBDNF), QTA021V (rAAV2-CAG-mBDNF-2A-eGFP), QTA022V (rAAV2-CAG-mBDNF-non-functional 2A-eGFP) and QTA023V (rAAV2-CAG-eGFP-2A-mBDNF). Three weeks later, expression of BDNF, TrkB receptors, eGFP and intracellular survival pathway activation was assessed by immunohistochemistry and Western blotting of retinal homogenates.
Results :
Constructs containing mBDNF and eGFP, separated by the viral-2A peptide linker, were successful processed in vivo to produce two transgenes which translocated to the correct intracellular compartments. Bands corresponding to mBDNF and eGFP were generated at the predicted molecular weights after injection of both QTA021V and QTA023V. QTA022V was not processed into two separate transgenes, as expected. Intravitreal injection of QTA020V showed efficient expression and processing with the amount of TrkB receptor increased 3-fold over non-injected naïve controls (1.14±0.19 vs 0.37±0.07; p<0.01, n=6). The amount of retinal BDNF measured was much greater in QTA020V treated eyes compared to controls (0.29±0.06 vs 0.00±0.00; p<0.001 n=6). Active phosphorylated-AKT and phosphorylated-ERK-1/2 proteins were also significantly increased, demonstrating activation of cell survival pathways after administration of QTA020V in vivo.
Conclusions :
QTA020V increased the density of both murine membrane-bound TrkB receptor and mBDNF within the retina and up-regulated cell survival pathways. Efficient cleavage of two transgenes was achieved when the viral-2A peptide linker was incorporated in the construct. QTA020V is predicted to provide potent RGC neuroprotection and further studies are currently underway to assess further the potential of QTA020V as a new treatment for glaucoma.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.