Sequencing-Guided Design of Genetically Encoded Small RNAs Targeting CAG Repeats for Selective Inhibition of Mutant Huntingtin [I]

Huntington’s disease (HD) is an incurable neurodegenerative disorder caused by genetic expansion of a CAG repeat sequence in one allele of the huntingtin (HTT) gene. Reducing expression of the mutant HTT (mutHTT) protein has remained a clear therapeutic goal but reduction of wild-type HTT (wtHTT) is undesirable as it compromises gene function and potential therapeutic efficacy. One promising allele- selective approach involves targeting the CAG repeat expansion with steric binding small RNAs bearing central mismatches. However, successful genetic encoding requires consistent placement of mismatches to the target within the small RNA guide sequence, which involves 5' processing precision by cellular enzymes. Here, we used small RNA sequencing to monitor processing precision of a limited set of CAG repeat- targeted small RNAs expressed from multiple scaffold contexts. Small RNA sequencing identified expression constructs with high guide strand 5' processing precision that also conferred promising allele-selective inhibition of mutHTT. However, mRNA-seq revealed varying degrees of transcriptome-wide off-target effects, including certain CAG repeat-containing mRNAs. These results support the continued investigation and optimization of genetically encoded repeat-targeted small RNAs for allele-selective HD gene therapy and underscore the value of sequencing methods to balance specificity with allele selectivity during the design and selection process. To identify processing of designed repeat shRNA/miRNAs in mammalian cell tissues, HEK 293T mammalian cells were transfected with repeat shRNA/miRNA encoding lentiviral plasmids. Sequence analysis was performed using pre-existing RNA seq analysis methods as well as in house excel spreadsheets for reconstructing read constructs. ImageJ software was used to evaluate transfection efficiency between transfected samples in order to normalize read abundance levels. Expression profiling using sRNAtoolbox and further analysis was used to evaluate transfected small RNAs and endogenous miRNA expression levels.

亨廷顿舞蹈症（Huntington’s disease, HD）是一种无法治愈的神经退行性疾病，其致病机制为亨廷顿（HTT）基因的其中一个等位基因上的CAG重复序列发生遗传扩增。降低突变型亨廷顿蛋白（mutHTT）的表达一直是明确的治疗靶点，但降低野生型亨廷顿蛋白（wtHTT）的表达并不可取，因其会损害基因功能并削弱潜在治疗效果。一种颇具前景的等位基因选择性治疗策略，是借助带有中心错配的空间结合型小RNA靶向CAG重复扩增区域。然而，要实现成功的遗传编码，需将错配精准且稳定地定位在小RNA引导序列的靶标区域内，这依赖于细胞酶对小RNA 5'端的精准加工。本研究通过小RNA测序，监测了由多种支架结构表达的一组有限的CAG重复序列靶向小RNA的加工精度。小RNA测序结果筛选出了一批引导链5'端加工精度高，且可有效实现等位基因选择性抑制mutHTT表达的表达构建体。但mRNA测序（mRNA-seq）结果显示，此类构建体存在不同程度的全转录组脱靶效应，其中包括对部分携带CAG重复序列的mRNA的影响。上述研究结果支持对遗传编码的重复序列靶向小RNA开展进一步研究与优化，以用于等位基因选择性HD基因治疗，同时凸显了测序方法在设计与筛选流程中平衡靶向特异性与等位基因选择性的重要价值。为鉴定哺乳动物细胞组织中设计的重复序列短发卡RNA/微RNA（shRNA/miRNA）的加工情况，本研究将编码重复序列shRNA/miRNA的慢病毒质粒转染至HEK 293T哺乳动物细胞中。序列分析采用已有的RNA测序分析方法，以及本实验室自制的Excel电子表格，用于重构测序读取的序列构建体。使用ImageJ软件评估各转染样本的转染效率，以对测序读取的丰度水平进行标准化校正。通过sRNAtoolbox进行表达谱分析并开展后续研究，以评估转染的小RNA及内源性miRNA的表达水平。