Shin Jun Wan, Hong Eun Pyo, Park Seri S, Choi Doo Eun, Zeng Sophia, Chen Richard Z, Lee Jong-Min
Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA 02114, USA.
Department of Neurology, Harvard Medical School, Boston, MA 02115, USA.
Mol Ther Methods Clin Dev. 2022 Aug 14;26:547-561. doi: 10.1016/j.omtm.2022.08.005. eCollection 2022 Sep 8.
Huntington's disease (HD) is caused by an expanded CAG repeat in huntingtin (). Since HD is dominant and loss of leads to neurological abnormalities, safe therapeutic strategies require selective inactivation of mutant . Previously, we proposed a concept of CRISPR-Cas9 using mutant-specific PAM sites generated by SNPs to selectively inactivate mutant . Aiming at revealing suitable targets for clinical development, we analyzed the largest HD genotype dataset to identify target PAM-altering SNPs (PAS) and subsequently evaluated their allele specificities. The gRNAs based on the PAM sites generated by rs2857935, rs16843804, and rs16843836 showed high levels of allele specificity in patient-derived cells. Simultaneous use of two gRNAs based on rs2857935-rs16843804 or rs2857935-rs16843836 produced selective genomic deletions in mutant and prevented the transcription of mutant mRNA without impacting the expression of normal counterpart or re-integration of the excised fragment elsewhere in the genome. RNA-seq and off-target analysis confirmed high levels of allele specificity and the lack of recurrent off-targeting. Approximately 60% of HD subjects are eligible for mutant-specific CRISPR-Cas9 strategies of targeting one of these three PAS in conjunction with one non-allele-specific site, supporting high applicability of PAS-based allele-specific CRISPR approaches in the HD patient population.
亨廷顿舞蹈症（HD）由亨廷顿蛋白中CAG重复序列扩增引起。由于HD是显性疾病，且亨廷顿蛋白缺失会导致神经异常，因此安全的治疗策略需要选择性地使突变型亨廷顿蛋白失活。此前，我们提出了一种利用单核苷酸多态性（SNP）产生的突变特异性原间隔序列临近基序（PAM）位点的CRISPR-Cas9概念，以选择性地使突变型亨廷顿蛋白失活。为了揭示适合临床开发的靶点，我们分析了最大的HD基因型数据集，以识别改变靶点PAM的SNP（PAS），并随后评估它们的等位基因特异性。基于rs2857935、rs16843804和rs16843836产生的PAM位点的引导RNA（gRNA）在患者来源的细胞中表现出高水平的等位基因特异性。同时使用基于rs2857935-rs16843804或rs2857935-rs16843836的两种gRNA，可在突变型亨廷顿蛋白中产生选择性基因组缺失，并阻止突变型亨廷顿蛋白mRNA的转录，而不会影响正常对应物的表达或切除片段在基因组其他位置的重新整合。RNA测序和脱靶分析证实了高水平的等位基因特异性以及缺乏反复脱靶现象。大约60%的HD患者符合将这三个PAS之一与一个非等位基因特异性位点结合的突变特异性CRISPR-Cas9策略，这支持了基于PAS的等位基因特异性CRISPR方法在HD患者群体中的高适用性。
