Development of the Gene Therapeutic Treatment to Mouse Senile Amyloidosis

由于治疗淀粉样变性的有效方法仅限于肝脏移植，因此，我们已经为开发淀粉样变性的基因治疗方法做了基础研究。1.小鼠老年性淀粉样变性的遗传学分析：在野生型B和淀粉样变性C型apoA-II杂合子R1.P1-APOA2^&lt；b/c&gt；这一发现提示了基因治疗方法的可能性。2.淀粉样变性小鼠肝细胞的原代培养：分离培养R1.P1-APOA2^c小鼠的肝细胞。3.基因治疗效果评价体系：由于淀粉样蛋白沉积通常在8-12个月龄后开始，我们试图建立一种在较短的时间内评价基因治疗效果的新体系。R1P1-APOA2c静脉注射淀粉样蛋白ApoAII后1个月诱导淀粉样蛋白沉积。在R1.P1-APOA2^&lt；b/c&gt；中，淀粉样蛋白沉积受到显著抑制。4.含野生型apoA-II基因的腺病毒：带有人5型腺病毒(Ad5)和强启动子单位的Cosmidcassette(PAdexCAwt)，CAG(巨细胞病毒增强子，鸡肌动蛋白启动子兔-珠蛋白Polya)由东京大学斋藤博士亲切提供。将野生型apoA-II基因(APOA2^c)亚克隆到粘粒的SWAL位点，与亲代腺病毒DNA-Tcp共转化293细胞。通过同源重组获得了几种野生型apoA-II基因的重组腺病毒。

Since, the effective treatment to amyloidosis is only the transplantation of the liver, we have done the basic studies for developing the gene therapeutic approach to amyloidosis. We used newly made congenic mice, R1.P1-Apoa2^c which are model mice of severe senile amyloidosis.1.Genetic analysis of mouse senile amyloidosis : In the congenic mice, R1.P1-Apoa2^<b/c> which are heterozygous for wild type B and amyloidogenic type C apoA-II,amyloid deposition was significantly slight. This finding suggested the possible availability of gene therapeutic approach. 2.Primary culture of the hepatic cell of the amyloidogenic mouse strain : Hepatic cells were isolated and cultured from R1.P1-Apoa2^c mice. 3.Evaluation system for the effectiveness of the gene therapy : Since the onset of amyloid deposition is usually after 8-12 month of age, we tried to develop a new system for evaluation the effect of gene therapy in relatively short period. Intravenous injection of amyloid fibrils AApoAII in R1.P1-Apoa2^c induced amyloid deposition at one month after injection. In R1.P1-Apoa2^<b/c>, amyloid deposition was significantly inhibited. 4.Adeno-virus with wild type apoA-II cDNA : Cosmid cassette (pAdexCAwt) which has human type 5 adenovirus (Ad5) and strong promoter unit, CAG (cytomegarovirus enhancer, chicken-actin promoter rabbit-globin polyA) was kindly provided by Dr.I.Saito in Tokyo University. Wild type apoA-II cDNA (Apoa2^c) was subcloned into Swal site of the cosmid and co-transformed with parental adeno virus DNA-TCP into the 293 cell. Several adeno viruses with wild type apoA-II cDNA produced by homologous recombination was selected.