Genome editing in HD iPS cells to reduce mutant and total Huntington expression

 描述(申请人提供)：亨廷顿病(HD)是一种致命的神经退行性疾病，发病于壮年，进展超过10-20年，产生运动异常、认知功能障碍和精神症状。HD是由亨廷顿蛋白(Huntingtin，Htt)基因蛋白编码区内CAG三核苷酸重复序列的显性扩张以及相应的皮质功能障碍和纹状体变性引起的。由于突变的HTT是致病因素，目前疾病干预的一个策略是减少HTT蛋白的产生。事实上，抑制RNA策略击倒突变型和野生型Htt等位基因已经在细胞和小鼠模型中显示出在减少HD相关表型方面的有效性。然而，考虑到正常HTT在神经发育和其他细胞功能中的重要作用，必须考虑降低Htt水平的有效性和降低Htt的副作用之间的平衡。出于这个原因，替代策略也侧重于选择性地降低mHTT水平，以减少潜在的副作用。最近，我们和其他人开发了HD患者来源的诱导多能干细胞(IPSCs)。这些细胞系可以分化为纹状体发育的成熟神经元，分化后的细胞表现出HD相关的表型，从而提供了一种系统，用于检查HD iPS细胞的“症状”进展以及这些表型在HTT降低后的可逆程度。在这里，我们建议使用现有HD iPSC系的基因组编辑来结构性或诱导性地表达以总HTT为目标或优先以突变等位基因为目标的RNAi。这种方法不同于等基因系。 其中，扩展的重复序列通过基因组编辑或同源重组被校正为野生型重复序列，以便在相同的遗传背景中验证依赖CAG的表型。在等基因背景下，没有疾病表型发生，因为校正的iPS细胞不再表达mHTT。相比之下，当考虑RNAi或ASO方法治疗HD时，患者在发育早期就不断表达扩大的重复突变，而改变这种慢性mHTT表达的影响的能力是RNAi或ASO所必需的。出现的问题是：1)CAG重复依赖性改变在mHTT背景下是否可以通过降低HTT来改变，2)一旦疾病表型明显，特定的mHTT表型是否可以通过完全或mHTT减少来改善，以及3)总体上降低HTT是否会产生后果，因为即使mHTT也可能具有在基因敲除策略下可能失去的功能。所提出的目标是：目标1。产生具有构成或诱导的Htt消音的高清iPS线。目的2.评价HD iPS细胞中总hTt降低与突变型hTt相比的结果。

 DESCRIPTION (provided by applicant): Huntington's disease (HD) is a fatal neurodegenerative disease which strikes in the prime of life and progresses over 10-20 years, producing movement abnormalities, cognitive dysfunction, and psychiatric manifestations. HD is caused by a dominant expansion of a CAG trinucleotide repeat tract within the protein-coding region of the Huntingtin (Htt) gene and corresponding cortical dysfunction and striatal degeneration. As mutant HTT is the disease causing agent, one current strategy for disease intervention is to reduce the production of the HTT protein. In fact, inhibitory RNA strategies which knock down both mutant and wild-type Htt alleles have shown effectiveness in cell and mouse models at reducing HD-related phenotypes. However, the balance between efficacy in reducing Htt levels and side effects from lowering Htt must be considered given the important roles for normal HTT in neurodevelopment and other cellular functions. For this reason, alternative strategies have also focused on selectively reducing mHtt levels to diminish potential side effects. Recently, we and others have developed HD patient-derived induced pluripotent stem cells (iPSCs). These lines can be differentiated to mature neurons specified for striatal development and differentiated cells display HD- related phenotypes, thereby providing a system in which to examine the progression of "symptoms" in HD iPS cells and the degree to which these phenotypes are reversible by HTT reduction. Here we propose to use genome editing of existing HD iPSC lines to constitutively or inducibly express RNAi's that target either total HTT or preferentially target the mutant allele. This approach is distinct from isogenic lines where the expanded repeat is corrected to wild type range repeats through genome editing or homologous recombination in order to validate CAG-dependent phenotypes in the same genetic background. In the isogenic context, no disease phenotypes occur as the corrected iPS cell no longer expresses mHTT. In contrast, when considering RNAi or ASO approaches for treatment of HD, patients continuously express the expanded repeat mutation beginning in early development and the ability to modify the impact of that chronic mHTT expression is what is required of that RNAi or ASO. Questions that emerge are 1) whether CAG repeat dependent changes are modifiable in a mHTT background through reduction of HTT, 2) whether specific mHTT phenotypes can be ameliorated by total or mHTT reduction once disease phenotypes are manifest and 3) whether reducing HTT in general has consequences as even mHTT is likely to have functions that could be lost following knockdown strategies. The proposed aims are: Aim 1. To generate HD iPS Lines with constitutive or inducible silencing of Htt. Aim 2. To evaluate the consequence of lowering total versus mutant HTT in HD iPS cells.