Experimental validation of gene therapy for ischemic stroke

Abstract

Aim. To develop a protocol of direct and cell-mediated gene therapy for ischemic stroke. Methods. Viral vector carrying green fluorescent protein (GFP) reporter gene was created on the basis of human adenovirus serotype 5 (Ad5). The umbilical blood supply was preserved according to instructions of Kazan State Medical Uuniversity Stem cell bank. Umbilical cord blood mononuclear cells were isolated in a ficoll density gradient by standard procedure and transduced with Ad5-GFP. Ischemic cerebral stroke in rats was caused by distal occlusion of the middle cerebral artery through trephination hole in a temporal bone under surgical microscope. Within four hours after modeling stroke in the anesthetized animals laminectomy was performed at the L4-L5 level, and (1) 0.9% sodium chloride solution, (2) Ad5-GFP and (3) umbilical cord blood mononuclear cells + Ad5-GFP were inserted intrathecally. Survival, targeted migration to the focus of neurodegeneration, the ability to synthesize recombinant protein and the effect of umbilical cord blood mononuclear cells on the infarction area were assessed using luminescent microscopy and morphometric analysis. Results. GFP expression in the area of the stroke was established 3 weeks after stroke modeling, both after intrathecal insertion of Ad5-GFP and after xenotransplantation of umbilical cord blood mononuclear cells Ad5-GFP transduced ex vivo. When comparing the areas of cerebral infarction 3 weeks after modeling the stroke, in animals from umbilical cord blood mononuclear cells + Ad5-GFP group the median of the infarction area was 47.4% less than in animals receiving isotonic saline solution. Conclusion. Umbilical cord blood mononuclear cells + Ad5-GFP after intrathecal insertion to animals with ischemic stroke, are capable of targeted migration to the neurodegeneration site as well as of recombinant protein synthesis; the results suggest the expediency of delivering therapeutic genes to ischemic zone via umbilical cord blood mononuclear cells overexpressing neurotrophic factors.