PDF(Pubmed)
Abstract：
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.
摘要：
Rett综合征是一种获得性进行性神经发育障碍，由X连锁MECP2基因的从头突变引起，该基因编码一种多效性蛋白，该蛋白充当全局转录调节因子和染色质修饰因子。Rett综合征主要影响杂合雌性，而受影响的雄性半合子很少存活。Rett综合征的基因疗法已被证明是具有挑战性的,因为需要对表达进行严格的调节,而过表达或过表达不足都是有毒的。MECP2与调控miRNA靶序列的异位表达已取得一定的成功，但是这种方法的持久性仍然未知。在这里，我们评估了基于无核酸酶同源重组（HR）的基因组编辑策略，以纠正MECP2基因中的突变。干细胞衍生的AAVHSC先前已被证明介导无缝和精确的基于HR的基因组编辑。我们测试了基于HR的基因组编辑纠正MECP2基因外显子3和4中致病性突变的能力，并恢复野生型序列，同时保留与MECP2表达相关的所有天然基因组调控元件。因此，有可能解决Rett综合征基因治疗中的一个重要问题。此外，因为突变是直接在基因组水平上编辑的，对于继承编辑基因的子代细胞，预计校正将是持久的。AAVHSCMECP2编辑载体被设计为与靶MECP2区域完全同源,并在外显子4的末端插入无启动子的Venus报道基因。在外显子3和4中携带突变的一组Rett细胞系中对AAVHSC编辑的评估表明成功校正和挽救编辑的MECP2基因的表达。编辑的Rett细胞的序列分析揭示了外显子3和外显子4两者中的突变的成功且准确的校正,并且允许HR交叉事件的定位。只有当交叉事件在5'和3'末端侧翼突变时才观察到成功的校正，但不是当两个交叉都发生在突变的上游或下游。重要的是,我们得出的结论是，在分析的每个Rett细胞系中，致病性突变都得到了成功纠正，证明了基于HR的基因组编辑的治疗潜力。
