Maria I Patrício1, Alun R Barnard1, Alexander L Green2, Matthew J During3, Arjune Sen4, Robert E MacLaren5. 1. Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford & NIHR Oxford Biomedical Research Centre, University of Oxford, Headley Way, Oxford OX3 9DU, UK; Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Headley Way, Oxford OX3 9DU, UK. 2. Oxford Epilepsy Research Group, Nuffield Department of Clinical Neurosciences, University of Oxford, Headley Way, Oxford OX3 9DU, UK; Nuffield Department of Surgery, University of Oxford, Headley Way, Oxford OX3 9DU, UK. 3. Ohio State University Wexner Medical Center, Columbus, OH 43210, USA. 4. Oxford Epilepsy Research Group, Nuffield Department of Clinical Neurosciences, University of Oxford, Headley Way, Oxford OX3 9DU, UK; Nuffield Department of Surgery, University of Oxford, Headley Way, Oxford OX3 9DU, UK. Electronic address: arjune.sen@ndcn.ox.ac.uk. 5. Nuffield Laboratory of Ophthalmology, Nuffield Department of Clinical Neurosciences, University of Oxford & NIHR Oxford Biomedical Research Centre, University of Oxford, Headley Way, Oxford OX3 9DU, UK; Oxford Eye Hospital, Oxford University Hospitals NHS Foundation Trust, Headley Way, Oxford OX3 9DU, UK. Electronic address: enquiries@eye.ox.ac.uk.
Abstract
PURPOSE: Epilepsy is a common neurological condition characterised by recurrent unprovoked seizures and often treatable with appropriate medication. However, almost 30% of cases are pharmacoresistant and while a proportion of these may be amenable to resective surgery, a gene therapy approach could be an attractive alternative option. Neuropeptide Y (NPY) has anticonvulsant and anti-epileptogenic properties in animal models of temporal lobe epilepsy when delivered by an adeno-associated viral (AAV) vector. Here we sought to demonstrate successful secretion of NPY from AAV-transduced human neuronal cells, which would be essential in planning any clinical trial. METHODS: A human neuroblastoma cell line (SH-SY5Y) was used to assess in vitro whether an AAV vector manufactured to clinical-grade protocols would be effective at transducing these cells to express NPY. Optimal transduction efficiency was first achieved with retinoic acid and tetradecanoylphorpol-13-acetate (TPA) treatment, prior to expose to AAV1-green fluorescent protein (GFP) reporter vector, AAV1-NPY therapeutic vector or sham treated with no vector. Levels of NPY in cell supernatants were determined using two antibody-based methods RESULTS: We found that the levels of NPY released into the cell culture media supernatant, and protein extracts of the cell pellet, were significantly higher following exposure to AAV1-NPY than when compared to either a control GFP reporter vector (AAV1-GFP) or sham treated controls. CONCLUSION: This first demonstration that an AAV-NPY construct can successfully transduce human neuronal cells supports the pre-clinical development of a clinical trial using AAV-based NPY for pharmacoresistant epilepsy.
PURPOSE:Epilepsy is a common neurological condition characterised by recurrent unprovoked seizures and often treatable with appropriate medication. However, almost 30% of cases are pharmacoresistant and while a proportion of these may be amenable to resective surgery, a gene therapy approach could be an attractive alternative option. Neuropeptide Y (NPY) has anticonvulsant and anti-epileptogenic properties in animal models of temporal lobe epilepsy when delivered by an adeno-associated viral (AAV) vector. Here we sought to demonstrate successful secretion of NPY from AAV-transduced human neuronal cells, which would be essential in planning any clinical trial. METHODS: A humanneuroblastoma cell line (SH-SY5Y) was used to assess in vitro whether an AAV vector manufactured to clinical-grade protocols would be effective at transducing these cells to express NPY. Optimal transduction efficiency was first achieved with retinoic acid and tetradecanoylphorpol-13-acetate (TPA) treatment, prior to expose to AAV1-green fluorescent protein (GFP) reporter vector, AAV1-NPY therapeutic vector or sham treated with no vector. Levels of NPY in cell supernatants were determined using two antibody-based methods RESULTS: We found that the levels of NPY released into the cell culture media supernatant, and protein extracts of the cell pellet, were significantly higher following exposure to AAV1-NPY than when compared to either a control GFP reporter vector (AAV1-GFP) or sham treated controls. CONCLUSION: This first demonstration that an AAV-NPY construct can successfully transduce human neuronal cells supports the pre-clinical development of a clinical trial using AAV-based NPY for pharmacoresistant epilepsy.
Authors: Noelia López-Sánchez; Alberto Garrido-García; Morgan Ramón-Landreau; Vanesa Cano-Daganzo; José M Frade Journal: Neurotherapeutics Date: 2021-11-11 Impact factor: 7.620