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 24-72 hours, 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.

A fusion of minicircle DNA and nanoparticle delivery technologies facilitates therapeutic genetic engineering of autologous canine olfactory mucosal cells

“…Compared with previous reports, the transfection levels we have obtained for cOMCs appear to be within ranges reported for viral methods: Carwardine et al 17 reported a maximum transduction efficiency of 34% at a multiplicity of infection (MOI) of 10, using chondroitinase ABC encoding lentiviral vectors and Ruitenberg et al 31 reported 100% transduction of rodent olfactory bulb-derived OECs with a reporter plasmid at an MOI of 50 and 100 using lentiviral and adenoviral vectors, respectively. Wu et al 32 transfected purified rodent olfactory bulb-derived OECs with a plasmid encoding NT-3 using a commercial lipofection agent, reporting a maximum transfection efficiency of 30% showing our techniques also compare favourably with other non-viral methods.…”

“…This means that the additional benefits provided by the MP platform, such as the use of bimodal nanoparticles for simultaneous gene delivery and imaging 27 could be safely exploited. Future work will deploy this therapy in vivo in clinical spinal injuries (including in canine trials), and explore the ability of this technical approach to deliver other genes involved in repair such as additional neurotherapeutic factors to promote growth, chondroitinase ABC to degrade the glial scar, 17,46 and VEGF to promote local angiogenesis. 47…”

“…Genetic engineering of OECs for release of neurotrophic factors at injury sites offers a potential strategy for improved regenerative outcomes; 13,14 for example, OECs genetically modified to secrete neurotrophin-3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF) show improved functionality post-transplantation versus non-engineered OECs. [15][16][17] Whilst work is ongoing to improve the safety of viral vector mediated bioengineering approaches, they can be associated with significant oncogenic 18 and inflammatory 19 risks to recipients.…”

Transplantation of canine olfactory ensheathing cells producing chondroitinase ABC promotes chondroitin sulphate proteoglycan digestion and axonal sprouting following spinal cord injury

“…The lentiviral transfer vector used to deliver the mammalian-compatible Proteus vulgaris ChABC gene [ 22 ] and the GFP gene to canine OECs was identical to the pRRL-CMV-ChABC-SFFV-GFP vector previously reported [ 37 ]. The control transfer vector pRRL-CMV-GFP expressed solely GFP.…”

“…Canine OECs can be safely collected from the nasal mucosa, cultured in vitro and autologously transplanted into spinal cord lesions [ 30 ]. We have previously demonstrated that these canine OECs can be genetically modified to produce the mammalian-modified form of ChABC in vitro [ 37 ]. We further show here that canine OECs can act as a vehicle for delivering active ChABC to the site of SCI in rats.…”