June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Design and characterisation of an AAV2 vector aiming to achieve long term modulation of BDNF signalling as a treatment for glaucoma
Author Affiliations & Notes
  • Peter S Widdowson
    QUETHERA Ltd, Cambridge, United Kingdom
  • Andrew Osborne
    Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom
    QUETHERA Ltd, Cambridge, United Kingdom
  • Alessia Tassoni
    Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom
  • Keith R Martin
    Department of Clinical Neurosciences, University of Cambridge, Cambridge, United Kingdom
    QUETHERA Ltd, Cambridge, United Kingdom
  • Footnotes
    Commercial Relationships   Peter Widdowson, Quethera Ltd (P), QUETHERA Ltd (E), QUETHERA Ltd (I); Andrew Osborne, QUETHERA Ltd (F); Alessia Tassoni, None; Keith Martin, QUETHERA Ltd (F), QUETHERA Ltd (I), QUETHERA Ltd (P), QUETHERA Ltd (C)
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 2569. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Peter S Widdowson, Andrew Osborne, Alessia Tassoni, Keith R Martin; Design and characterisation of an AAV2 vector aiming to achieve long term modulation of BDNF signalling as a treatment for glaucoma. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2569.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
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.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×