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

 描述（由申请人提供）：亨廷顿病（HD）是一种致命的神经退行性疾病，在生命的黄金时期发作，并在10-20年内进展，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（Htt）基因的蛋白编码区内CAG三核苷酸重复序列的显性扩增以及相应的皮质功能障碍和纹状体变性引起的。由于突变体HTT是致病因子，目前的一种疾病干预策略是减少HTT蛋白的产生。事实上，敲低突变体和野生型Htt等位基因的抑制性RNA策略在细胞和小鼠模型中显示出减少HD相关表型的有效性。然而，鉴于正常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水平的功效和降低Htt的副作用之间的平衡。出于这个原因，替代策略也集中在选择性地降低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（iPSC）。这些细胞系可以分化为特定用于纹状体发育的成熟神经元，并且分化的细胞显示HD相关的表型，从而提供了一种系统，在该系统中检查HD iPS细胞中“症状”的进展以及这些表型通过HTT减少可逆的程度。在这里，我们提出使用现有HD iPSC系的基因组编辑来组成性地或诱导性地表达靶向总HTT或优先靶向突变等位基因的RNAi。这种方法不同于等基因系 其中通过基因组编辑或同源重组将扩增的重复序列校正为野生型范围重复序列，以验证相同遗传背景中的CAG依赖性表型。在同基因背景下，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或阿索方法用于治疗HD时，患者在早期发育中开始持续表达扩展的重复突变，并且改变慢性mHTT表达的影响的能力是RNAi或阿索所需的。出现的问题是1）CAG重复依赖性变化是否可在mHTT背景下通过HTT的减少来改变，2）一旦疾病表型显现，特定mHTT表型是否可通过总的或mHTT的减少来改善，以及3）通常减少HTT是否具有后果，因为甚至mHTT也可能具有在敲除策略后可能丧失的功能。拟议的目标是：目标1。产生具有组成型或诱导型Htt沉默的HD iPS系。目标2.评价降低HD iPS细胞中总HTT与突变HTT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.