Abstract:
We aimed to produce a mouse model of spinocerebellar ataxia type 3 (SCA3) using the mouse blood-brain barrier (BBB)-penetrating adeno-associated virus (AAV)-PHP.B. Four-to-five-week-old C57BL/6 mice received injections of high-dose (2.0 × 1011 vg/mouse) or low-dose (5.0 × 1010 vg/mouse) AAV-PHP.B encoding a SCA3 causative gene containing abnormally long 89 CAG repeats [ATXN3(Q89)] under the control of the ubiquitous chicken β-actin hybrid (CBh) promoter. Control mice received high doses of AAV-PHP.B encoding ATXN3 with non-pathogenic 15 CAG repeats [ATXN3(Q15)] or phosphate-buffered saline (PBS) alone. More than half of the mice injected with high doses of AAV-PHP.B encoding ATXN3(Q89) died within 4 weeks after the injection. No mice in other groups died during the 12-week observation period. Mice injected with low doses of AAV-PHP.B encoding ATXN3(Q89) exhibited progressive motor uncoordination starting 4 weeks and a shorter stride in footprint analysis performed at 12 weeks post-AAV injection. Immunohistochemistry showed thinning of the molecular layer and the formation of nuclear inclusions in Purkinje cells from mice injected with low doses of AAV-PHP.B encoding ATXN3(Q89). Moreover, ATXN3(Q89) expression significantly reduced the number of large projection neurons in the cerebellar nuclei to one third of that observed in mice expressing ATXN3(Q15). This AAV-based approach is superior to conventional methods in that the required number of model mice can be created simply by injecting AAV, and the expression levels of the responsible gene can be adjusted by changing the amount of AAV injected. Moreover, this method may be applied to produce SCA3 models in non-human primates.
摘要:
我们旨在使用小鼠血脑屏障(BBB)穿透腺相关病毒(AAV)-PHP.B产生脊髓小脑共济失调3型(SCA3)的小鼠模型。四至五周大的C57BL/6小鼠接受了高剂量(2.0×1011vg/小鼠)或低剂量(5.0×1010vg/小鼠)AAV-PHP的注射。B编码SCA3致病基因,该基因包含在普遍存在的鸡β-肌动蛋白杂种(CBh)启动子控制下的异常长的89个CAG重复序列[ATXN3(Q89)]。对照小鼠接受高剂量的AAV-PHP。B编码具有非致病性CAG重复的ATXN3[ATXN3(Q15)]或单独的磷酸盐缓冲盐水(PBS)。超过一半的小鼠注射高剂量的AAV-PHP。编码ATXN3的B(Q89)在打针后4周内逝世亡。在12周的观察期间,其他组中没有小鼠死亡。小鼠注射低剂量的AAV-PHP。编码ATXN3(Q89)的B表现出从4周开始的进行性运动不协调,并且在AAV注射后12周时进行足迹分析的跨步较短。免疫组织化学显示,注射低剂量AAV-PHP的小鼠的浦肯野细胞中分子层变薄并形成核内含物。B编码ATXN3(Q89)。此外,ATXN3(Q89)表达将小脑核中大投射神经元的数量显著减少至表达ATXN3(Q15)的小鼠中观察到的数量的三分之一。这种基于AAV的方法优于常规方法,因为只需注射AAV即可创建所需数量的模型小鼠。并且可以通过改变注射的AAV的量来调节负责基因的表达水平。此外,该方法可用于在非人灵长类动物中产生SCA3模型。