Large-Scale Chondroitin Sulfate Proteoglycan Digestion with Chondroitinase Gene Therapy Leads to Reduced Pathology and Modulates Macrophage Phenotype following Spinal Cord Contusion Injury

Abstract

Chondroitin sulfate proteoglycans (CSPGs) inhibit repair following spinal cord injury. Here we use mammalian-compatible engineered chondroitinase ABC (ChABC) delivered via lentiviral vector (LV-ChABC) to explore the consequences of large-scale CSPG digestion for spinal cord repair. We demonstrate significantly reduced secondary injury pathology in adult rats following spinal contusion injury and LV-ChABC treatment, with reduced cavitation and enhanced preservation of spinal neurons and axons at 12 weeks postinjury, compared with control (LV-GFP)-treated animals. To understand these neuroprotective effects, we investigated early inflammatory changes following LV-ChABC treatment. Increased expression of the phagocytic macrophage marker CD68 at 3 d postinjury was followed by increased CD206 expression at 2 weeks, indicating that large-scale CSPG digestion can alter macrophage phenotype to favor alternatively activated M2 macrophages. Accordingly, ChABC treatment in vitro induced a significant increase in CD206 expression in unpolarized monocytes stimulated with conditioned medium from spinal-injured tissue explants. LV-ChABC also promoted the remodelling of specific CSPGs as well as enhanced vascularity, which was closely associated with CD206-positive macrophages. Neuroprotective effects of LV-ChABC corresponded with improved sensorimotor function, evident as early as 1 week postinjury, a time point when increased neuronal survival correlated with reduced apoptosis. Improved function was maintained into chronic injury stages, where improved axonal conduction and increased serotonergic innervation were also observed. Thus, we demonstrate that ChABC gene therapy can modulate secondary injury processes, with neuroprotective effects that lead to long-term improved functional outcome and reveal novel mechanistic evidence that modulation of macrophage phenotype may underlie these effects.

Canine olfactory ensheathing cells from the olfactory mucosa can be engineered to produce active chondroitinase ABC

“…One group has taken the approach of thermo-stabilizing ChABC and achieved sustained delivery of the enzyme for up to 6 weeks using a hydrogelmicrotube scaffold (65). Other groups have pursued gene therapy as a means to improve sustained enzyme delivery (22,23,28,29,62,63). An alternative approach to disabling the inhibitory CSPGs is to target the CSPG receptor present on neurons: protein tyrosine phosphatase σ (PTPσ) (66).…”

“…Digestion of CSPGs with the bacterial enzyme chondroitinase ABC (ChABC), following local delivery to the spinal cord, has led to axon regeneration, plastic neuronal rearrangements and functional recovery following section or crush injury in laboratory animal SCI models (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Encouragingly, these findings have also been found using clinically relevant contusive injury rodent models (22,23) and large animal models such as cats and squirrel monkeys (19,24,25). Despite these advances, a major limitation for the use of ChABC in humans is the rapid decay of enzymatic activity at body temperature, within 24 to 72 hours (26).…”

“…ChABC is likely to form an essential component of any combination therapy because of its pleotrophic actions that compliment most other SCI therapies. It promotes plasticity, is neuroprotective and also has an antiinflammatory action by promoting the polarization of macrophages to the antiinflammatory M2 type (23).…”

“…This sequence has been incorporated into both adeno-associated viral vectors and lentiviral vectors as a delivery system of the modified ChABC gene into mammalian cells (22,23,28,29). Lentiviral vectors transduce host cells efficiently and this results in long-term expression of the incorporated transgenes.…”

“…They also are less immunogenic than other viral delivery systems such as adenoviral-based vectors (30,31). In vivo studies testing viral transduction of cells within the spinal cord following intraparenchymal injection of a lentiviral vector containing the mammalian form of the ChABC gene resulted in widespread CSPGs digestion over several spinal segments and this persisted for at least 2 months (22,23).…”