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 treatmentin vitroinduced 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.

Lentiviral vectors are very efficient at transducing dividing and quiescent cells, which makes them highly useful tools for genetic analysis and gene therapy. Traditionally this efficiency was considered dependent on provirus integration in the host cell genome; however, recent results have challenged this view. So called integration-deficient lentiviral vectors (IDLVs) can be produced through the use of integrase mutations that specifically prevent proviral integration, resulting in the generation of increased levels of circular vector episomes in transduced cells. These lentiviral episomes lack replication signals and are gradually lost by dilution in dividing cells, but are stable in quiescent cells. Compared to integrating lentivectors, IDLVs have a greatly reduced risk of causing insertional mutagenesis and a lower risk of generating replication-competent recombinants (RCRs). IDLVs can mediate transient gene expression in proliferating cells, stable expression in nondividing cells in vitro and in vivo, specific immune responses, RNA interference, homologous recombination (gene repair, knock-in, and knock-out), site-specific recombination, and transposition. IDLVs can be converted into replicating episomes, suggesting that if a clinically applicable system can be developed they would also become highly appropriate for stable transduction of proliferating tissues in therapeutic applications.