Gene Therapy Reduction of Radiotherapy Esophagitis

描述（由申请人提供）：非小细胞肺癌放化疗的主要剂量限制性毒性是辐照性食管炎。本应用旨在证明电离辐照通过微环境细胞持续产生活性氧（ROS）限制了辐照后食管干细胞的修复性植入，以及食道内mnsod -质粒脂质体（PL）（抗氧化剂）基因治疗改善毒性的机制是通过稳定抗氧化剂池。我们已经证明，食道内给药MnSOD-PL降低了单次或分次放疗对小鼠食管的毒性，减少了辐射诱导的谷胱甘肽和硫醇耗损、脂质过氧化，并提高了通过两侧群体（SP）细胞分选或7天预板技术分离的静脉注射食管干细胞的存活率。我们建议利用MnSOD-PL抗氧化治疗通过减少微环境中ROS的产生来促进植根。我们将采用三种方法来测量辐照诱导的毒性ROS对骨髓源性食管干细胞移植的影响：1)供体男性C57BL/6J （FYDR）细胞的克隆同源重组（HR）；2)供体细胞与受体食管融合；3)原位和移植食管干细胞的凋亡。第一个具体目的是验证G418r LacZ+ (B6.129S7-Gt(ROSA)26Sor/J)雄性小鼠骨髓源连续移植食管祖细胞归巢和增殖到受辐照雌性受体食管的假设，从而产生细胞剂量依赖性的辐照保护，并与G418r LacZ+集落形成鳞状细胞祖细胞的体外恢复相关。第二个特定目的是验证辐照诱导的ROS（特别是过氧亚硝酸盐）在辐照雌性小鼠食管微环境中诱导供体C57BL/6J雄性（FYDR）骨髓SP细胞（克隆黄色病灶）的HR，细胞与受体食管细胞融合和凋亡的假设。第三个具体目的是验证假说，即在ROSA和FYDR骨髓细胞移植模型中，MnSOD-PL处理对辐照食管抗氧化储备的恢复，稳定了骨髓干细胞移植。方法包括将来自雄性C57BL/6J、FYDR和选择性G418抗性LacZ （ROSA）雄性供体的食道和造血干细胞亚群植入受辐射受体C57BL/6J雌性GFP+小鼠中，进行细胞分选、FISH、体外和体内抗氧化剂生化测定，以及直接杀伤和旁观杀伤测定。这些研究将阐明辐照食管微环境诱导移植骨髓源性上皮干细胞损伤的分子机制，并确定新的治疗干预靶点。

DESCRIPTION (provided by applicant): A major dose limiting toxicity of chemoradiotherapy of non-small cell carcinoma of the lung is irradiation esophagitis. This application seeks to prove that ionizing irradiation limits reparative stem cell engraftment of the irradiated esophagus through continuous reactive oxygen species (ROS) production by cells of the microenvironment, and that the mechanism of amelioration of toxicity by intraesophageal MnSOD-Plasmid Liposome (PL) (antioxidant) gene therapy is by stabilizing the antioxidant pool. We have demonstrated that intra-esophageal administration of MnSOD-PL decreases the toxicity of single fraction or fractionated radiotherapy to the mouse esophagus, decreases irradiation-induced glutathione and thiol depletion, lipid peroxidation, and enhance the survival of intravenously injected esophageal stem cells isolated by either side population (SP) cell sorting or 7-day pre-plate technique. We propose to utilize MnSOD-PL antioxidant therapy to enhance engraftment by reducing ROS production by the microenvironment. We will use three assays for measuring irradiation-induced toxic ROS effects on bone marrow derived esophageal stem cell engraftment: 1) clonal homologous recombination (HR) in donor male C57BL/6J (FYDR) cells; 2) fusion of donor cells with recipient esophagus; and 3) apoptosis of both resident and engrafted esophageal stem cells. The first specific aim tests the hypothesis that homing and proliferation of bone marrow origin serially transplantable esophageal progenitor cells from G418r LacZ+ (B6.129S7-Gt(ROSA)26Sor/J) male mice to the irradiated female recipient esophagus results in cell dose dependent irradiation protection, and correlates with recovery of G418r LacZ+ colony forming squamous cell progenitors in vitro. The second specific aim tests the hypothesis that irradiation-induced ROS, specifically peroxynitrite, in the microenvironment of the irradiated female mouse esophagus induces HR in donor C57BL/6J male (FYDR) marrow SP cells quantitated as clonal yellow foci, cell fusion with recipient esophageal cells and apoptosis. The third specific aim tests the hypothesis that restoration of antioxidant reserves in the irradiated esophagus by MnSOD-PL treatment, in both ROSA and FYDR marrow cell transplantation models, stabilizes marrow stem cell engraftment. Methods include engraftment of donor esophageal and hematopoietic stem cell subsets from male C57BL/6J, FYDR, and selectable G418 resistant LacZ (ROSA) male donors into irradiated recipient C57BL/6J female GFP+ mice, cell sorting, FISH, biochemical measurements of antioxidants in vitro and in vivo, and assays for direct and bystander killing. These studies should elucidate the molecular mechanism of induction of damage to engrafting marrow origin, epithelial stem cells by the irradiated esophageal microenvironment and identify new targets for therapeutic intervention.