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

HD iPS 细胞中的基因组编辑可减少突变体和总亨廷顿表达

• 批准号: 8970040
• 负责人: Leslie Michels Thompson
• 金额: $ 19.31万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-15 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8970040
• 关键词: Address; Adverse effects; Alleles; CAG repeat; Cell Line; Cell model; Cell physiology; Cells; Chronic; Clustered Regularly Interspaced Short Palindromic Repeats; Cognitive; Communities; Corpus striatum structure; Development; Disease; Disease Progression; Effectiveness; Employee Strikes; Equilibrium; Functional disorder; Genes; Genetic; Human; Huntington Disease; Impaired cognition; Inherited; Intervention; Knowledge; Life; Mediating; MicroRNAs; Microscopy; Molecular; Movement; Mutate; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Differentiation; Neurons; Open Reading Frames; Pathogenesis; Pathology; Patients; Phenotype; Production; Proteins; RNA; RNA Interference; Research; Resources; Robotics; Role; Site; Specific qualifier value; Staging; Symptoms; System; Technology; Therapeutic; Trinucleotide Repeats; base; disease phenotype; genome editing; homologous recombination; human Huntingtin protein; induced pluripotent stem cell; innovation; insight; knock-down; mouse model; mutant; nervous system disorder; neurodevelopment; neuronal survival; prevent; promoter; public health relevance; small molecule; transcriptomics

 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.

 描述（由申请人提供）：亨廷顿舞蹈症 (HD) 是一种致命的神经退行性疾病，在生命的壮年时期发病，并持续 10-20 年，导致运动异常、认知功能障碍和精神表现。 HD 是由亨廷顿蛋白 (Htt) 基因蛋白质编码区内 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。由于突变型 HTT 是致病因子，目前疾病干预的一种策略是减少 HTT 蛋白的产生。事实上，敲除突变型和野生型 Htt 等位基因的抑制性 RNA 策略已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 水平的功效与降低 Htt 的副作用之间的平衡。因此，替代策略也集中在选择性降低 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纹状体发育特异的成熟神经元，并且分化的细胞显示HD相关表型，从而提供一个系统来检查HD iPS细胞中“症状”的进展以及这些表型通过HTT还原可逆转的程度。在这里，我们建议使用现有 HD iPSC 系的基因组编辑来组成型或诱导型表达靶向总 HTT 或优先靶向突变等位基因的 RNAi。这种方法与等基因系不同 其中通过基因组编辑或同源重组将扩展的重复序列校正为野生型范围重复序列，以便在相同遗传背景下验证 CAG 依赖性表型。在同基因背景下，由于校正后的 iPS 细胞不再表达 mHTT，因此不会出现疾病表型。相比之下，当考虑用 RNAi 或 ASO 方法治疗 HD 时，患者从早期发育开始就持续表达扩展的重复突变，而改变慢性 mHTT 表达影响的能力正是 RNAi 或 ASO 所需要的。出现的问题是：1）在 mHTT 背景下，CAG 重复依赖性变化是否可以通过减少 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；3）总体上减少 HTT 是否会产生后果，因为即使是 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. 生成具有 Htt 组成型或诱导型沉默的 HD iPS 系。目标 2. 评估 HD iPS 细胞中降低总 HTT 与突变体 HTT 的结果。