Developing combination strategies for the treatment of spinal cord injury utilizing ex vivo gene therapy and alginate encapsulation

Spinal cord injury (SCI) affects between 10,000 and 20,000 people each year in the United States. Most of these patients are left with motor and/or sensory dysfunction that limits their recovery. At the present time there is no treatment to repair the injured spinal cord or restore lost function. In the last decade, preclinical studies with transplants of stem cells and non-neural primary cells in combination with gene therapy have offered much promise for treatment of SCI. One of these therapies, ex vivo gene therapy utilizing primary fibroblasts genetically modified to secrete brain-derived neurotrophic factor (BDNF) or neurotrophic-factor-3 (NT-3), have been shown to be a successful strategy for stimulating regeneration, rescuing axotomized brainstem neurons, and eliciting behavioral recovery in adult rodent models of acute SCI. These studies were performed by grafting cells that secrete only one neurotrophin, which targeted a specific spinal tract that was injured. The goal of this thesis is to explore the capacity for combining therapeutic approaches utilizing genetically engineered fibroblasts to maximize neuron survival, regeneration and behavioral recovery for the treatment of acute and chronic SCI. Chapter 2 introduces this theme by showing that retroviral producer cell titers could be significantly increased by the addition of an inhibitor of histone deacetylase (trichostatin A) to an infection protocol. Chapters 3 and 4 show that grafting a combination of BDNF and NT-3 producing fibroblasts after a 6-week delay into the injured adult rat SCI site partially rescues axotomized Red nucleus neurons from loss and atrophy, provides a permissive environment for growth of chronically injured host axons, but elicits only modest recovery of function. Chapters 5 and 6 show that BDNF-producing fibroblasts that have been encapsulated in a semi-permeable alginate poly-L-ornithine membrane survive in culture for at least two months, remain viable after freezing in liquid nitrogen, create a permissive environment for host axons growth after grafting into the acutely injured spinal cord, and elicit behavioral recovery in tests of vertical forelimb exploration (cylinder test) and hindlimb function (rope test) in the absence of immune suppression. These results, utilizing a combination approach, demonstrate the diverse way in which engineered fibroblasts and alginate encapsulation can benefit the acutely and chronically injured adult spinal cord and provide evidence for their usefulness in treatment of human SCI.