Redesigned chondroitinase ABC degrades inhibitory chondroitin sulfate proteoglycans in vitro and in vivo in the stroke-injured rat brain

Abstract

Injuries to the central nervous system, such as stroke and traumatic spinal cord injury, result in an aggregate scar that both limits tissue degeneration and inhibits tissue regeneration. The aggregate scar includes chondroitin sulfate proteoglycans (CSPGs), which impede cell migration and axonal outgrowth. Chondroitinase ABC (ChASE) is a potent yet fragile enzyme that degrades CSPGs, and thus may enable tissue regeneration. ChASE37, with 37-point mutations to the native enzyme, has been shown to be more stable than ChASE, but its efficacy has never been tested. To answer this question, we investigated the efficacy of ChASE37 first in vitro using human cell-based assays and then in vivo in a rodent model of stroke. We demonstrated ChASE37 degradation of CSPGs in vitro and the consequent cell adhesion and axonal sprouting now possible using human induced pluripotent stem cell (hiPSC)-derived neurons. To enable prolonged release of ChASE37 to injured tissue, we expressed it as a fusion protein with a Src homology 3 (SH3) domain and modified an injectable, carboxymethylcellulose (CMC) hydrogel with SH3-binding peptides (CMC-bp) using inverse electron-demand Diels−Alder chemistry. We injected this affinity release CMC-bp/SH3-ChASE37 hydrogel epicortically to endothelin-1 stroke-injured rats and confirmed bioactivity via degradation of CSPGs and axonal sprouting in and around the lesion. With CSPG degradation shown both in vitro by greater cell interaction and in vivo with local delivery from a sustained release formulation, we lay the foundation to test the potential of ChASE37 and its delivery by local affinity release for tissue regeneration after stroke.