Abstract
Purpose :
Our aim is to develop a gene therapy for glaucoma based on modulation of brain-derived neurotrophic factor (BDNF) signalling. AAV-mediated BDNF expression has previously been shown to protect retinal ganglion cells in animal models of glaucoma, but long-term efficacy is compromised by TrkB receptor down-regulation. We designed a gene construct coding for both murine TrkB and BDNF, increasing receptor and neurotrophin expression to aid long-term RGC survival.
Methods :
To enhance efficacy and fit our construct within the 4.7kb packaging limit of an rAAV2 vector the endogenous BDNF signal peptide was modified and the coding sequence for proBDNF removed to leave a mature BDNF (mBDNF) transgene. Murine TrkB and mBDNF coding sequences were joined using the viral-2A peptide sequence to create a single transgene, QTA020V, controlled by the CAG promoter. Construct components were studied in vitro in HEK293 cells using lipofectamine transduced DNA plasmids. BDNF production and secretion was measured by Western blot and ELISA. Correct cleavage of the single transgene into TrkB and BDNF was assessed by Western blot and immunocytochemistry. Effects on neuroprotective pathways downstream of murine TrkB receptor was also tested.
Results :
Our novel BDNF signal peptide increased the concentration of BDNF secreted from HEK293 cells compared to the endogenous sequence (extracellular BDNF 73.1±7.3 vs 23.5±1.7ng/mL; P<0.001). The precursor protein was effectively processed following transfection to yield murine TrkB receptor and mBDNF. Testing of the dual construct revealed enhanced activation of RGC survival pathways AKT (2.36±0.13-fold) and ERK1/2 (30.22±1.86-fold) compared to controls (P<0.05 n=6), with activation comparable to the addition of 200ng/mL BDNF to cells over-expressing TrkB alone. BDNF released from HEK293 cells transfected with the dual construct was increased from 0.45±0.07 to 1.30±0.17 ng/mL (P<0.001 n=8) when ANA-12 (10μM) a TrkB receptor blocker was added, providing evidence of autocrine signalling.
Conclusions :
QTA020V is a novel gene therapy designed to provide long term activation of the BDNF/TrkB signalling pathway. Intracellular processing of the single gene construct into constitutive mBDNF and murine TrkB was demonstrated to be highly efficient in vitro resulting in activation of down-stream cellular survival and anti-apoptotic pathways.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.