Axonal Regeneration Using Transplants of Genetically Engineered Fibroblasts in Spinal Cord Injury

Philadelphia, PA, USA

INTRODUCTION: In a recent publication from our institution (J. Neuroscience, June, 1999), regeneration of the rubrospinal tract was demonstrated in an adult rat cervical spinal cord injury model with recovery of forelimb function. This was achieved through transplantation of cell cultures of fibroblasts genetically modified to secrete NT3 (neurotrophin-3) and BDNF (brain derived neurotrophic factor), using ex-vivo gene therapy, into a hemi-section model. The purpose of the current study was to achieve regeneration across a complete cord transection defect.

METHODS: 21 adult rats underwent mid-thoracic laminectomy and complete spinal cord transection, creating an approximately 3 mm defect. The defect was filled with a carefully titrated number of genetically modified fibroblasts which were engineered using retroviral vectors to secrete neurotrophic factors (NT-3, and BDNF), which are known to enhance axonal regeneration. Animals received modified fibroblasts suspended in Vitrogen (a collagen based carrier); powdered gelfoam, platelet gel, and without matrix. Post-operatively, rats were functionally tested using the BBB test for locomotion. Rats also received cyclosporin A immunosuppression post-operatively. At two months rats were sacrificed. Graft site and adjacent spinal cord tissue was harvested and serially sectioned for histologic analysis. Immunofluorescent mouse antibody labeling for neurofiliment protein (RT97) and serotonin was also carried out.

RESULTS: Data for a complete spinal cord transection model supports prior published data for cervical hemisections. Robust regeneration of adult CNS axons was observed traversing the spinal cord defect and for several segments distal to the injury. Overall transplanted fibroblast survival was high in each group. A trend towards more axonal regeneration was noted in groups which received a higher titrated number of modified fibroblasts. Some functional recovery was noted in rats receiving transplants versus controls by increases in BBB score by 2-3 grades.

SUMMARY: By providing specific nerve growth factors using gene therapy, regeneration of CNS axons in adult rats across a complete spinal cord transection was achieved. Regeneration using ex-vivo gene therapy is a significant step in the development of treatments for spinal cord injury in humans. Future studies will focus on combining ex-vivo gene therapy with pharmacological and rehabilitative treatments to produce the greatest possible behavioral recovery, first in experimental models, and later clinically.