无核酸精确基因组编辑可纠正与雷特综合征相关的 MECP2 基因突变

IF 4.9 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Swati Bijlani, Ka Ming Pang, Lakshmi V. Bugga, Sampath Rangasamy, Vinodh Narayanan, Saswati Chatterjee
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引用次数: 0

摘要

雷特综合征是一种获得性进行性神经发育障碍,由 X 连锁 MECP2 基因的新突变引起。雷特综合征主要影响杂合子雌性患者,而受影响的男性半杂合子患者很少存活。Rett 综合征的基因治疗具有挑战性,因为需要严格的表达调控,过度表达或表达不足都会产生毒性。结合调控 miRNA 目标序列的 MECP2 异位表达取得了一些成功,但这种方法的持久性仍然未知。在这里,我们评估了一种基于无核酸酶同源重组(HR)的基因组编辑策略,以纠正MECP2基因的突变。干细胞衍生的AAVHSCs先前已被证明能介导无缝、精确的基于HR的基因组编辑。我们测试了基于HR的基因组编辑纠正MECP2基因第3和第4外显子中致病突变的能力,并在保留与MECP2表达相关的所有原生基因组调控元件的同时恢复野生型序列,从而有可能解决Rett综合征基因治疗中的一个重要问题。此外,由于突变是直接在基因组水平上进行编辑的,因此预计这种校正将是持久的,后代细胞将继承编辑后的基因。AAVHSC MECP2 编辑载体的设计与目标 MECP2 区域完全同源,并在外显子 4 的末端插入了无启动子的 Venus 报告基因。在一组外显子 3 和 4 发生突变的 Rett 细胞系中对 AAVHSC 编辑进行的评估表明,编辑后的 MECP2 基因的表达得到了成功的纠正和挽救。对编辑过的 Rett 细胞进行序列分析后发现,第 3 和第 4 外显子中的突变都得到了成功而准确的校正,并能绘制出 HR 交叉事件的图谱。只有当突变的5′端和3′端两侧都有交叉事件时,才能观察到成功的校正,而当两个交叉事件都完全发生在突变的上游或下游时,则不能观察到成功的校正。重要的是,我们得出结论,在分析的每个 Rett 株系中,致病突变都被成功纠正,这证明了基于 HR 的基因组编辑具有治疗潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Nuclease-free precise genome editing corrects MECP2 mutations associated with Rett syndrome
Rett syndrome is an acquired progressive neurodevelopmental disorder caused by de novo mutations in the X-linked MECP2 gene which encodes a pleiotropic protein that functions as a global transcriptional regulator and a chromatin modifier. Rett syndrome predominantly affects heterozygous females while affected male hemizygotes rarely survive. Gene therapy of Rett syndrome has proven challenging due to a requirement for stringent regulation of expression with either over- or under-expression being toxic. Ectopic expression of MECP2 in conjunction with regulatory miRNA target sequences has achieved some success, but the durability of this approach remains unknown. Here we evaluated a nuclease-free homologous recombination (HR)-based genome editing strategy to correct mutations in the MECP2 gene. The stem cell-derived AAVHSCs have previously been shown to mediate seamless and precise HR-based genome editing. We tested the ability of HR-based genome editing to correct pathogenic mutations in Exons 3 and 4 of the MECP2 gene and restore the wild type sequence while preserving all native genomic regulatory elements associated with MECP2 expression, thus potentially addressing a significant issue in gene therapy for Rett syndrome. Moreover, since the mutations are edited directly at the level of the genome, the corrections are expected to be durable with progeny cells inheriting the edited gene. The AAVHSC MECP2 editing vector was designed to be fully homologous to the target MECP2 region and to insert a promoterless Venus reporter at the end of Exon 4. Evaluation of AAVHSC editing in a panel of Rett cell lines bearing mutations in Exons 3 and 4 demonstrated successful correction and rescue of expression of the edited MECP2 gene. Sequence analysis of edited Rett cells revealed successful and accurate correction of mutations in both Exons 3 and 4 and permitted mapping of HR crossover events. Successful correction was observed only when the mutations were flanked at both the 5′ and 3′ ends by crossover events, but not when both crossovers occurred either exclusively upstream or downstream of the mutation. Importantly, we concluded that pathogenic mutations were successfully corrected in every Rett line analyzed, demonstrating the therapeutic potential of HR-based genome editing.
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