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Design of a Novel Gene Therapy Construct to Achieve Sustained Brain-Derived Neurotrophic Factor Signaling in Neurons.

Published version
Peer-reviewed

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Authors

Osborne, Andrew 
Wang, Aiden XZ 
Tassoni, Alessia 
Widdowson, Peter S 
Martin, Keith R 

Abstract

Brain-derived neurotrophic factor (BDNF) acting through the tropomyosin-related receptor-B (TrkB) is an important signaling system for the maintenance and survival of neurons. Gene therapy using either recombinant adeno-associated virus (AAV) or lentiviral vectors can provide sustained delivery of BDNF to tissues where reduced BDNF signaling is hypothesized to contribute to disease pathophysiology. However, elevation in BDNF at target sites has been shown to lead to a downregulation of TrkB receptors, thereby reducing the effect of chronic BDNF delivery over time. A novel gene sequence has been designed coding both the ligand (BDNF) and the TrkB receptor in a single transgene separated by a short viral-2A sequence. The single transgene is efficiently processed intracellularly in vitro and in vivo to yield the two mature proteins, which are then independently transported to their final cellular locations: TrkB receptors to the cell surface, and BDNF contained within secretory vesicles. To accommodate the coding sequences of both BDNF and TrkB receptors within the narrow confines of the AAV vectors (4.7 kb pairs), the coding region for the pro-domain of BDNF was removed and the signal peptide sequence modified to improve production, intracellular transport, and secretion of mature BDNF (mBDNF). Intracellular processing and efficacy was shown in HEK293 cells and SH-SY5Y neuroblastoma cells using plasmid DNA and after incorporating the TrkB-2A-mBDNF into an AAV2 vector. Increased BDNF/TrkB-mediated intracellular signaling pathways were observed after AAV2 vector transfection while increased TrkB phosphorylation could be detected in combination with neuroprotection from hydrogen peroxide-induced oxidative stress. Correct processing was also shown in vivo in mouse retinal ganglion cells after AAV2 vector administration to the eye. This novel construct is currently being investigated for its efficacy in animal models to determine its potential to progress to human clinical studies in the future.

Description

Keywords

adeno-associated viral vector, brain-derived neurotrophic factor, gene therapy, tropomyosin-related receptor kinase-B, Animals, Brain-Derived Neurotrophic Factor, Dependovirus, Genetic Therapy, HEK293 Cells, Humans, Hydrogen Peroxide, Ligands, Membrane Glycoproteins, Mice, Neurons, Oxidative Stress, Phosphorylation, Protein Sorting Signals, Receptor, trkB, Retinal Ganglion Cells

Journal Title

Hum Gene Ther

Conference Name

Journal ISSN

1043-0342
1557-7422

Volume Title

29

Publisher

Mary Ann Liebert Inc
Sponsorship
Cambridge Eye Trust (unknown)
Fight for Sight (5061/5062)