Local delivery of stabilized chondroitinase ABC degrades chondroitin sulfate proteoglycans in stroke-injured rat brains

Abstract

Central nervous system (CNS) injuries, such as stroke and spinal cord injuries, result in the formation of a proteoglycan-rich glial scar, which acts as a barrier to axonal regrowth and limits the regenerative capacity of the CNS. Chondroitinase ABC (ChABC) is a potent bacterial enzyme that degrades the chondroitin sulfate proteoglycan (CSPG) component of the glial scar and promotes tissue recovery; however, its use is significantly limited by its inherent instability at physiological temperatures. Here, we demonstrate that ChABC can be stabilized using site-directed mutagenesis and covalent modification with poly(ethylene glycol) chains (i.e. PEGylation). Rosetta protein structure modeling was used to screen >20,000 single point mutations, and four potentially stabilizing mutations were tested in vitro. One of the mutations, N1000G (asparagine ➔ glycine at residue 1000), significantly improved the long-term activity of the protein, doubling its functional half-life. PEGylation of this ChABC mutant inhibited unfolding and aggregation and resulted in prolonged bioactivity with a 10-fold increase in activity compared to the unmodified protein after two days. Local, affinity-controlled release of the modified protein (PEG-N1000G-ChABC) was achieved by expressing it as a fusion protein with Src homology 3 (SH3) and delivering the protein from a methylcellulose hydrogel modified with SH3 binding peptides. This affinity-based release strategy provided sustained PEG-N1000G-ChABC-SH3 release over several days in vitro. Direct implantation of the hydrogel delivery vehicle containing stabilized PEG-N1000G-ChABC-SH3 onto the rat brain cortex in a sub-acute model of stroke resulted in significantly reduced CSPG levels in the penumbra of 49% at 14 and 40% at 28 days post-injury compared to animals treated with the vehicle alone.