1499. Bioengineered SMaRT Human Neural Stem Cells to Degrade Scar and Enhance Regeneration in Chronic Spinal Cord Injury

Authors: Christopher Ahuja, MD; Mohamad Khazaei; Zijian Lou; Priscilla Chan; Sohanthen Udayashankar; Nayaab Punjani; William Luong; Suyue Lyu; Jian Wang; Michael Fehlings (Ajax, Canada)

Introduction:

Human induced pluripotent stem cell-derived neural stem cells (hiPS-NSCs) are an exciting strategy for traumatic spinal cord injury (SCI) as they have the capacity to replace neural circuits, remyelinate denuded axons and provide trophic support. Unfortunately, most individuals are in the chronic injury phase where dense perilesional chondroitin-sulfate proteoglycan (CSPG) scarring significantly impairs neurite outgrowth and cell migration. Scar-modifying enzymes can enhance NSC-mediated recovery, however, nonspecific intrathecal administration causes off-target effects. We aimed to generate a novel, genetically-engineered line of hiPS-NSCs, termed Spinal Microenvironment Modifying and Regenerative Therapeutic (SMaRT) cells, capable of locally expressing a scar-degrading enzyme to enhance recovery.

Methods:

Using non-viral techniques, a scar degrading enzyme was genetically integrated into hiPS-NSCs under a human promoter and a monoclonal line was generated by fluorescence activated cell sorting. Enzyme expression and activity was extensively characterized in vitro by biochemical and cell culture assays. T-cell deficient rats (N=60) with chronic (8-week) C6-7 clip-contusion injuries were randomized to receive: (1)NSCs, (2)SMaRT enzyme-expressing NSCs, (3)vehicle control, or (4)sham surgery. Behavioural assessments were completed to 40 weeks post-injury.

Results:

The scar-degrading enzyme and fluorescent reporter are robustly expressed by the transgenic SMaRT cells. Importantly, SMaRT cells retain key human NSC characteristics. The expressed enzyme appropriately degrades human CSPGs and allows neurons to extend into CSPG-rich regions in vitro. While blinded behavioural analyses are ongoing, an interim histologic analysis shows grafted human cells extending remarkably long (medulla to mid-thoracic) axons through rodent white matter at 8 weeks post-transplant. The graft further evolves by 32 weeks post-transplant demonstrating more numerous, thinner, and longer processes with positive staining for mature neuron marker, NF200.

Conclusion:

This work provides exciting proof-of-concept data that genetically-engineered SMaRT cells can degrade CSPGs in vitro and that human NSC grafts can form long axonal processes in the challenging chronic cervical SCI niche.