title: Canine olfactory ensheathing cells from the olfactory mucosa can be engineered to produce active chondroitinase ABC.
abstract: A multitude of factors must be overcome following spinal cord injury (SCI) in order to achieve clinical improvement in patients. It is thought that by combining promising therapies these diverse factors could be combatted with the aim of producing an overall improvement in function. Chondroitin sulphate proteoglycans (CSPGs) present in the glial scar that forms following SCI present a significant block to axon regeneration. Digestion of CSPGs by chondroitinase ABC (ChABC) leads to axon regeneration, neuronal plasticity and functional improvement in preclinical models of SCI. However, the enzyme activity decays at body temperature within 24-72h, limiting the translational potential of ChABC as a therapy. Olfactory ensheathing cells (OECs) have shown huge promise as a cell transplant therapy in SCI. Their beneficial effects have been demonstrated in multiple small animal SCI models as well as in naturally occurring SCI in canine patients. In the present study, we have genetically modified canine OECs from the mucosa to constitutively produce enzymatically active ChABC. We have developed a lentiviral vector that can deliver a mammalian modified version of the ChABC gene to mammalian cells, including OECs. Enzyme production was quantified using the Morgan-Elson assay that detects the breakdown products of CSPG digestion in cell supernatants. We confirmed our findings by immunolabelling cell supernatant samples using Western blotting. OECs normal cell function was unaffected by genetic modification as demonstrated by normal microscopic morphology and the presence of the low affinity neurotrophin receptor (p75(NGF)) following viral transduction. We have developed the means to allow production of active ChABC in combination with a promising cell transplant therapy for SCI repair.
authors.affiliation: University of Bristol, School of Veterinary Sciences, Regenerative Medicine Laboratory, Biomedical Science Building, University Walk, Bristol BS8 1TD, United Kingdom. Electronic address: darren.carwardine@bristol.ac.uk.
authors.forename: Darren
authors.initials: D
authors.lastname: Carwardine
authors.affiliation: University of Bristol, School of Clinical Sciences, Regenerative Medicine Laboratory, Biomedical Science Building, University Walk, Bristol BS8 1TD, United Kingdom. Electronic address: l.wong@bristol.ac.uk.
authors.forename: Liang-Fong
authors.initials: LF
authors.lastname: Wong
authors.affiliation: University of Cambridge, Department of Clinical Neurosciences, Cambridge Centre for Brain Repair, E.D. Adrian Building, Forvie Site, Robinson Way, Cambridge CB2 0PY, United Kingdom. Electronic address: jf108@cam.ac.uk.
authors.forename: James W
authors.initials: JW
authors.lastname: Fawcett
authors.affiliation: University of Cambridge, Department of Physiology Development and Neuroscience, Anatomy Building, Downing St, Cambridge CB2 3DY, United Kingdom. Electronic address: emm1@cam.ac.uk.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Muir
authors.affiliation: University of Bristol, School of Veterinary Sciences, Langford House, Langford, North Somerset BS40 5DU, United Kingdom. Electronic address: nicolas.granger@bristol.ac.uk.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Granger
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D015153:Blotting, Western; D019765:Chondroitin ABC Lyase / Q000235:genetics / Q000378:metabolism*; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism; D004285:Dogs; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D016086:Lentivirus / Q000235:genetics; D009831:Olfactory Mucosa / Q000166:cytology* / Q000201:enzymology* / Q000637:transplantation; D011514:Proteus vulgaris / Q000201:enzymology / Q000235:genetics; D017475:Receptors, Nerve Growth Factor / Q000378:metabolism; D013119:Spinal Cord Injuries / Q000628:therapy; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D011508:Chondroitin Sulfate Proteoglycans; D017475:Receptors, Nerve Growth Factor; D049452:Green Fluorescent Proteins; D019765:Chondroitin ABC Lyase
keywords: Chondroitin sulphate proteoglycan; Chondroitinase ABC; Gene therapy; Olfactory ensheathing cells; Olfactory mucosa; Spinal cord injury