Novel Ex Vivo Gene Therapy for Spinal Cord Injury

DESCRIPTION (provided by applicant): There are approximately 10,000 traumatic spinal cord injuries in the United States each year. The average age of injury is 31 years. The majority (90%) of individuals survive and live near-normal life spans. Cost of treating these injuries is very high and does not lead to complete recovery. Spinal cord injury can result in progressive axonal degeneration, variable amounts of neuronal loss, and functional impairment. Effective therapies have to be designed to not only minimize the loss of tissue due to secondary processes but also to promote axonal growth and functional regeneration. We are developing cell-based delivery of proteins (neurotrophic factors) that help in regeneration of neurons. Towards testing this therapy we propose the following specific aims: Specific Aim 1: To perform dose response studies with Sertoli cells. Our current data suggest that Sertoli cells alone had a significant effect on functional recovery and rate of locomotor recovery of spinal cord injured animals. This effect was observed at early time points and plateaued thereafter. One interpretation is that there simply are not enough cells to sustain the effect overtime. To address that possibility we will inject varying amounts of Sertoli cells into the acutely injured spinal cord. Sertoli cells will be implanted into white matter caudal to the site of injury, within 30 min of the injury, using a stereotaxic device. Lesion development will be monitored by in vivo magnetic resonance imaging (MRI). The intent is to correlate the temporal profile of lesion progression with locomotor recovery, as defined by a battery of neurologic tests that evaluate coordinated locomotion, hind limb paw position during locomotion, and performance on an inclined grid. To our knowledge this is the first study to use MRI to determine the extent to which lesion progression correlates with neurologic impairment. Animals will be euthanized at 42 days post injury and the spinal cords will be subject to histological assessment of white matter and quantification of NT-3. Specific Aim 2: To determine if delayed implantation of cells following injury results in improved cell survival and functional recovery. In the above studies we have implanted cells in the acutely traumatized spinal cord. The acutely injured spinal cord is characterized by prominent oxidative injury and pronounced proteolytic activity. Thus, survival of Sertoli cells may be challenged by these adverse conditions. We therefore propose to delay the time of cell implantation to 3 days and 7 days post injury. These time points coincide with wound healing including angiogenesis, events that may define an environment that is more receptive to implantation of cells. Lesion development will be monitored by in vivo magnetic resonance imaging (MRI) and correlated with motor function. Animals will be euthanized at 42 days post injury. The cords will be subjected to immuno- and histochemistry and assessment of NT-3 activity. Emphasis will be on quantifying the extent of residual white matter, glial scar formation, and assessment of the long descending serotonergic fiber tracts. Together, these anatomical and behavioral outcomes will determine the extent to which treatment alters wound healing and promotes plasticity and functional recovery. Specific Aim 3: To isolate and characterize human Sertoli cells for therapeutic development as delivery agents. Human Sertoli cells will be isolated from tissues, propagated in culture, and carefully characterized with regard to their expression of surface proteins including follicle stimulating hormone receptor (FSHr) and androgen binding protein (ABP). Gamma-glutamyl transpeptidase (GGT); a marker for Sertoli cells, we be quantified using a standard enzyme assay. Once characterized, cells will be infected with recombinant adenovirus expressing NT-3 and the levels of NT-3 production will be determined by ELISA. Mixed lymphocyte reactions will be performed in the presence of virally infected and uninfected human Sertoli cells, to determine their immunosuppressive ability.