Pre-clinical study of gene therapy against refractory diseases in animals.

本研究的目的是研究兽医领域针对肿瘤和免疫异常的基因治疗的进展。在本研究期间，开展了1)肝细胞生长因子(HGF)基因治疗的基础研究，2)犬P53腺病毒载体的构建及其对犬肿瘤细胞的作用，3)猫免疫缺陷病毒(FIV)的基因治疗。1)HGF基因治疗狗和猫的肝肾功能不全：克隆了猫和猫的HGF基因，并与肿瘤转化生长因子(TGF)一起检测了它们在犬各种肝病中的表达。2)猫、猫肿瘤的基因治疗：观察P53抑癌基因在犬多种肿瘤中的异常及中心体的异常情况。接下来，我们构建了表达犬P53基因的腺病毒载体，并检测了其对犬肿瘤细胞株的杀伤作用。3)FIV感染的基因治疗：我们建立了一种新的检测血浆中FIV RNA的方法，并研究了血浆病毒RNA载量与自然感染FIV猫预后的关系。我们还研究了FIV感染细胞的凋亡机制和克隆的猫科肿瘤坏死因子受体。此外，还克隆了猫血管内皮细胞生长因子(VEGFR)基因，以开发针对FIV感染的新的基因治疗方法。

The object of this study is the development of gene therapy against tumors and immune-abnormalities in veterinary field. In this research period, the study of 1) basic research of gene therapy by hepatocyte growth factor (HGF), 2) construction of adenovirus vector containing canine p53 and its effects for canine tumor cell lines, 3) gene therapy for and by feline immunodeficiency virus (FIV), have been conducted.1) HGF-gene therapy for liver and renal insufficiency in dogs and cats : Feline and canine HGF cDNA were cloned and their expressions were examined in various liver diseases in dogs together with tumor transforming growth factor (TGF). Furthermore, feline and canine HGF cDNA was inserted into expression plasmid and their efficient gene delivery method was examined.2) Gene therapy for tumors in dogs and cats : Aberrations of the p53 tumor suppressor gene in various tumors in dogs was investigated together with the abnormalities of centrosome. We next constructed adenovirus vector expressing canine p53 which was cloned in our laboratory and examined its cytocidal effects on canine tumor cell lines. Furthermore, feline IL-18 cDNA was cloned and expression plasmid for canine CTLA-4 fusion protein to enhance anti-tumor immunity.3) Gene therapy for FIV infection : We developed the novel method to quantify FIV RNA in plasma and an association of plasma viral RNA load with prognosis in cats naturally infected with FIV was investigated. We also investigated the mechanism of apoptosis of FIV-infected cells and cloned feline TNF receptors. Furthermore, feline vascular endotherial growth factor (VEGF) cDNA was cloned to develop new gene therapy against FIV infection.