PMID- 29466871 OWN - NLM STAT- MEDLINE DCOM- 20190624 LR - 20190624 IS - 1557-7422 (Electronic) IS - 1043-0342 (Print) IS - 1043-0342 (Linking) VI - 29 IP - 7 DP - 2018 Jul TI - Design of a Novel Gene Therapy Construct to Achieve Sustained Brain-Derived Neurotrophic Factor Signaling in Neurons. PG - 828-841 LID - 10.1089/hum.2017.069 [doi] AB - 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. FAU - Osborne, Andrew AU - Osborne A AD - 1 John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge , Cambridge, United Kingdom. AD - 2 Quethera Ltd. , Babraham Research Campus, Cambridge, United Kingdom. FAU - Wang, Aiden X Z AU - Wang AXZ AD - 1 John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge , Cambridge, United Kingdom. FAU - Tassoni, Alessia AU - Tassoni A AD - 1 John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge , Cambridge, United Kingdom. FAU - Widdowson, Peter S AU - Widdowson PS AD - 2 Quethera Ltd. , Babraham Research Campus, Cambridge, United Kingdom. FAU - Martin, Keith R AU - Martin KR AD - 1 John van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge , Cambridge, United Kingdom. AD - 2 Quethera Ltd. , Babraham Research Campus, Cambridge, United Kingdom. AD - 3 Cambridge NIHR Biomedical Research Centre , Cambridge, United Kingdom. AD - 4 Eye Department, Addenbrooke's Hospital , Cambridge, United Kingdom. AD - 5 Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge , Cambridge, United Kingdom . LA - eng GR - Wellcome Trust/United Kingdom PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20180402 PL - United States TA - Hum Gene Ther JT - Human gene therapy JID - 9008950 RN - 0 (Brain-Derived Neurotrophic Factor) RN - 0 (Ligands) RN - 0 (Membrane Glycoproteins) RN - 0 (Protein Sorting Signals) RN - 7171WSG8A2 (BDNF protein, human) RN - BBX060AN9V (Hydrogen Peroxide) RN - EC 2.7.10.1 (Receptor, trkB) RN - EC 2.7.10.1 (tropomyosin-related kinase-B, human) SB - IM MH - Animals MH - Brain-Derived Neurotrophic Factor/administration & dosage/*genetics MH - Dependovirus/genetics MH - *Genetic Therapy MH - HEK293 Cells MH - Humans MH - Hydrogen Peroxide/toxicity MH - Ligands MH - Membrane Glycoproteins/genetics MH - Mice MH - Neurons/*drug effects/pathology MH - Oxidative Stress/*drug effects/genetics MH - Phosphorylation MH - Protein Sorting Signals/genetics MH - Receptor, trkB/administration & dosage/*genetics MH - Retinal Ganglion Cells/drug effects/pathology PMC - PMC6066195 OTO - NOTNLM OT - adeno-associated viral vector OT - brain-derived neurotrophic factor OT - gene therapy OT - tropomyosin-related receptor kinase-B COIS- P.S.W. and K.R.M. have a financial interest in Quethera Ltd., a company working to develop gene therapy approaches to neurodegenerative diseases. A.O., A.T., and K.R.M. have received grant support from Quethera Ltd. A.X.Z.W. has no disclosures. EDAT- 2018/02/23 06:00 MHDA- 2019/06/25 06:00 PMCR- 2018/07/01 CRDT- 2018/02/23 06:00 PHST- 2018/02/23 06:00 [pubmed] PHST- 2019/06/25 06:00 [medline] PHST- 2018/02/23 06:00 [entrez] PHST- 2018/07/01 00:00 [pmc-release] AID - 10.1089/hum.2017.069 [pii] AID - 10.1089/hum.2017.069 [doi] PST - ppublish SO - Hum Gene Ther. 2018 Jul;29(7):828-841. doi: 10.1089/hum.2017.069. Epub 2018 Apr 2.