Huntington’s disease (HD) is an autosomal-dominant neurodegenerative genetic disorder caused by CAG repeat expansion in exon 1 of the HTT gene. Expression of the mutant gene results in the production of a neurotoxic polyglutamine (polyQ)-expanded huntingtin (Htt) protein. Clinical trials of knockdown therapy of mutant polyglutamine-encoding HTT mRNA in Huntington’s disease (HD) have begun. To measure HTT mRNA knockdown effectiveness in human cells, we utilized a fluorescent hybridization imaging agent specific to the region encompassing the human HTT mRNA initiation codon. We designed, synthesized, purified, and characterized Cal560-spacer-peptide nucleic acid (PNA)-spacer-IGF1 tetrapeptides. The human HTT PNA 12mer complement was CATGGCGGTCTC, while the rat htt equivalent 12mer contained the sequence CATGaCGGcCTC, with two bases differing from the human sequence. The cyclized IGF1 tetrapeptide fragment d(CSKC) that promotes IGF1 receptor-mediated endocytosis was bonded to the C-terminus. We tested the reliability of HTT mRNA imaging with Cal560-spacer-peptide nucleic acid (PNA)-spacer-IGF1 tetrapeptides in human embryonic kidney (HEK) 293T cells that express endogenous HTT and IGF1 receptor. By qPCR, we quantitated HTT mRNA in HEK293T cells with and without HTT mRNA knockdown by three different siRNAs. By confocal fluorescence imaging, we quantitated the accumulation of fluorescent HTT hybridization agent in the same cells. A rat homologue differing from the human sequence by two bases showed negligible fluorescence. qPCR indicated 86 ± 5% knockdown of HTT mRNA by the most effective siRNA. Similarly, Cal560-HTT PNA-peptide fluorescence intensity indicated 69 ± 6% reduction in HTT mRNA. We concluded that the fluorescence hybridization method correlates with the established qPCR method for quantitating HTT mRNA knockdown by siRNA in HEK293T cells, with a Pearson correlation coefficient of 0.865 for all three siRNA sequences. These results will enable real time imaging and quantitation of HTT mRNA in animal models of HD.

中文翻译：

---

Huntingtin mRNA敲低的荧光成像

亨廷顿舞蹈病（HD）是常染色体显性遗传神经退行性遗传疾病，由HTT基因第1外显子的CAG重复扩增引起。突变基因的表达导致产生神经毒性的聚谷氨酰胺（polyQ）扩展的亨廷顿蛋白（Htt）。亨廷顿氏病（HD）中编码突变型聚谷氨酰胺的HTT mRNA的敲除疗法的临床试验已经开始。为了测量人类细胞中HTT mRNA的敲低效果，我们利用了一种荧光杂交显像剂，该试剂对包含人类HTT mRNA起始密码子的区域具有特异性。我们设计，合成，纯化和表征了Cal560-间隔物-肽核酸（PNA）-间隔物-IGF1四肽。人类HTTPNA 12mer补体为CATGGCGGTCTC，而大鼠htt等效12mer包含序列CATG a CGG c CTC，其中两个碱基与人序列不同。促进IGF1受体介导的内吞作用的环化IGF1四肽片段d（CSKC）与C末端结合。我们在表达内源性HTT和IGF1受体的人胚胎肾脏（HEK）293T细胞中，用Cal560-间隔肽核酸（PNA）-间隔物-IGF1四肽测试了HTT mRNA成像的可靠性。通过qPCR，我们定量了有和没有HTT的HEK293T细胞中的HTT mRNA通过三种不同的siRNA进行mRNA敲低。通过共聚焦荧光成像，我们定量了荧光HTT杂交剂在相同细胞中的积累。与人类序列相差两个碱基的大鼠同源物显示出可忽略的荧光。qPCR表明，最有效的siRNA可使HTT mRNA的敲除率达86±5％。类似地，Cal560 - HTT PNA-肽的荧光强度表明HTT mRNA降低了69±6％。我们得出的结论是，荧光杂交方法与已建立的定量HTT的qPCR方法相关siRNA在HEK293T细胞中的mRNA敲低，所有三个siRNA序列的皮尔逊相关系数均为0.865。这些结果将使高清动物模型中HTT mRNA的实时成像和定量成为可能。