PDF(Pubmed)
Abstract：
The pathological huntingtin (HTT) trinucleotide repeat underlying Huntington disease (HD) continues to expand throughout life. Repeat length correlates both with earlier age at onset (AaO) and faster progression, making slowing its expansion an attractive therapeutic approach. Genome-wide association studies have identified candidate variants associated with altered AaO and progression, with many found in DNA mismatch repair (MMR)-associated genes. We examine whether lowering expression of these genes affects the rate of repeat expansion in human ex vivo models using HD iPSCs and HD iPSC-derived striatal medium spiny neuron-enriched cultures. We have generated a stable CRISPR interference HD iPSC line in which we can specifically and efficiently lower gene expression from a donor carrying over 125 CAG repeats. Lowering expression of each member of the MMR complexes MutS (MSH2, MSH3, and MSH6), MutL (MLH1, PMS1, PMS2, and MLH3), and LIG1 resulted in characteristic MMR deficiencies. Reduced MSH2, MSH3, and MLH1 slowed repeat expansion to the largest degree, while lowering either PMS1, PMS2, or MLH3 slowed it to a lesser degree. These effects were recapitulated in iPSC-derived striatal cultures where MutL factor expression was lowered. CRISPRi-mediated lowering of key MMR factor expression to levels feasibly achievable by current therapeutic approaches was able to effectively slow the expansion of the HTT CAG tract. We highlight members of the MutL family as potential targets to slow pathogenic repeat expansion with the aim to delay onset and progression of HD and potentially other repeat expansion disorders exhibiting somatic instability.
摘要：
作为亨廷顿病（HD）基础的病理性亨廷顿（HTT）三核苷酸重复序列在整个生命中持续扩展。重复长度与较早的发病年龄（AaO）和较快的进展相关，使减缓其扩张成为一种有吸引力的治疗方法。全基因组关联研究已经确定了与AaO和进展改变相关的候选变体。在DNA错配修复（MMR）相关基因中发现了许多。我们使用HDiPSC和HDiPSC衍生的纹状体培养基多刺状神经元富集培养物，研究了降低这些基因的表达是否会影响人类离体模型中重复扩增的速率。我们已经产生了稳定的CRISPR干扰HDiPSC系,其中我们可以特异性地和有效地降低来自携带超过125个CAG重复的供体的基因表达。降低MMR复合物MutS（MSH2，MSH3和MSH6）的每个成员的表达，MutL（MLH1、PMS1、PMS2和MLH3），和LIG1导致特征性MMR缺陷。MSH2、MSH3和MLH1的减少最大程度地减缓了重复膨胀，而降低PMS1，PMS2或MLH3的速度则较小。这些作用在iPSC衍生的纹状体培养物中被概括,其中MutL因子表达降低。CRISPRi介导的关键MMR因子表达降低至通过当前治疗方法可行可实现的水平能够有效地减缓HTTCAG束的扩张。我们强调MutL家族的成员是减缓致病性重复扩展的潜在目标，目的是延迟HD的发作和进展以及其他可能表现出躯体不稳定性的重复扩展障碍。
