256. Novel Therapeutic Strategy for Ischemic Stroke by Bone Marrow Stromal Cells and Ex Vivo HGF and FGF2 Genes Transfer with Replication-Incompetent HSV-1 Vector
Authors: Naosuke Nonoguchi, Ming-Zhu Zhao, Naokado Ikeda, Yoshihito Miki, Daisuke Furutama, Fumiharu Kimura, Mari Dezawa, Hiroshi Funakoshi, Robert S. Coffin, Shin-ichi Miyatake, Toshihiko Kuroiwa
Journal: Molecular Therapy (2006)

The bone marrow stromal cells (MSCs), including the primitive pluriopotent mesenchymal stem cells (PMSCs) and the multipotent adult progenitor cells (MAPCs), are attractive targets for cell and gene therapy for various central nervous system disorders.Material and method: For gene modification of MSCs, we used a replication-incompetent HSV-1 vector "1764/4-", triple-deletion mutant of the ICP4, ICP34.5, and VP16. This vector has very high ability of gene transduction to MSC, 70-75% at MOI 10 and 95% at MOI 50, and its expression can carry on at least one month at high level. Two kinds of vectors expressing HGF and FGF-2 were prepared for this study. FGF-2 vector contains the signal sequence of Interleukin-2 (IL-2ss) ahead of FGF-2 gene to enhance its extracellular secretion, because native FGF2 gene don't have the excretion signal. Native MSCs, HGF-MSCs, IL2ss+FGF-2-MSCs and PBS were administered directly into the lesioned brain 24 hours after transient middle cerebral artery occlusion (MCAO) of adult Wister rats.Results: At day 14, each gene modified MSC group showed significant improvement in modified neurological severity score (mNSS) and apparent decreasing of infarct volume compared with native MSC or sham-operated group. (p<0.05). HGF and FGF-2 expression in vivo were strongly kept through the experimental period. In HGF modified MSC-treated group, the MAP-2 positive cell% tends to increase and the TUNEL positive cell% decrease in the ischemic boundary zone (IBZ). The transdifferentiation of transplanted MSCs to central nervous system cell phenotypes was also checked. Most of the transdifferentiated phenotype was GFAP- positive, and the Nestin-positive cells were scarcely able to find out in the host brain.Discussion and Conclusion: By using the replication- incompetent HSV-1 vector, MSCs can be efficiently engineered to secrete a series of various cytokines in large quantities and in the long term in vitro and in vivo that could potentially treat the ischemic stroke of the brain. HGF and FGF-2 gene-transferred MSCs, achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native MSCs in rat MCAO model. It would be appeared that the main mechanism of these therapeutic effects is the neuroprotection (anti-apoptotic effect) in IBZ by various abundant cytokines secreted from gene engineered MSCs. We will present our detail data of the study and the perspective of this therapeutic strategy. The bone marrow stromal cells (MSCs), including the primitive pluriopotent mesenchymal stem cells (PMSCs) and the multipotent adult progenitor cells (MAPCs), are attractive targets for cell and gene therapy for various central nervous system disorders. Material and method: For gene modification of MSCs, we used a replication-incompetent HSV-1 vector "1764/4-", triple-deletion mutant of the ICP4, ICP34.5, and VP16. This vector has very high ability of gene transduction to MSC, 70-75% at MOI 10 and 95% at MOI 50, and its expression can carry on at least one month at high level. Two kinds of vectors expressing HGF and FGF-2 were prepared for this study. FGF-2 vector contains the signal sequence of Interleukin-2 (IL-2ss) ahead of FGF-2 gene to enhance its extracellular secretion, because native FGF2 gene don't have the excretion signal. Native MSCs, HGF-MSCs, IL2ss+FGF-2-MSCs and PBS were administered directly into the lesioned brain 24 hours after transient middle cerebral artery occlusion (MCAO) of adult Wister rats. Results: At day 14, each gene modified MSC group showed significant improvement in modified neurological severity score (mNSS) and apparent decreasing of infarct volume compared with native MSC or sham-operated group. (p<0.05). HGF and FGF-2 expression in vivo were strongly kept through the experimental period. In HGF modified MSC-treated group, the MAP-2 positive cell% tends to increase and the TUNEL positive cell% decrease in the ischemic boundary zone (IBZ). The transdifferentiation of transplanted MSCs to central nervous system cell phenotypes was also checked. Most of the transdifferentiated phenotype was GFAP- positive, and the Nestin-positive cells were scarcely able to find out in the host brain. Discussion and Conclusion: By using the replication- incompetent HSV-1 vector, MSCs can be efficiently engineered to secrete a series of various cytokines in large quantities and in the long term in vitro and in vivo that could potentially treat the ischemic stroke of the brain. HGF and FGF-2 gene-transferred MSCs, achieved the remarkable amelioration of neurological symptoms and apparent decreasing of infarct volume comparison with native MSCs in rat MCAO model. It would be appeared that the main mechanism of these therapeutic effects is the neuroprotection (anti-apoptotic effect) in IBZ by various abundant cytokines secreted from gene engineered MSCs. We will present our detail data of the study and the perspective of this therapeutic strategy.