Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare disease caused by recessive mutations in the TYMP gene, which encodes the enzyme thymidine phosphorylase (TP). In this study, the efficient integration of a TYMP transgene into introns of the Tymp and Alb loci of hepatocytes in a murine model of MNGIE was achieved by the coordinated delivery and activity of CRISPR/Cas9 and a TYMP cDNA. CRISPR/Cas9 was delivered either as mRNA using lipid nanoparticle (LNP) or polymeric nanoparticle, respectively, or in an AAV2/8 viral vector; the latter was also used to package the TYMP cDNA. Insertion of the cDNA template downstream of the Tymp and Alb promoters ensured transgene expression. The best in vivo results were obtained using LNP carrying the CRISPR/Cas9 mRNAs. Treated mice showed a consistent long-term (1 year) reduction in plasma nucleoside (thymidine and deoxyuridine) levels that correlated with the presence of TYMP mRNA and functional enzyme in liver cells. In mice with an edited Alb locus, the transgene produced a hybrid Alb-hTP protein that was secreted, with supraphysiological levels of TP activity detected in the plasma. Equivalent results were obtained in mice edited at the Tymp locus. Finally, some degree of gene editing was found in animals treated only with AAV vectors containing the DNA templates, in the absence of nucleases, although there was no impact on plasma nucleoside levels. Overall, these results demonstrate the feasibility of liver-directed genome editing in the long-term correction of MNGIE, with several advantages over other methods.

中文翻译：

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线粒体神经胃肠道脑肌病小鼠模型中基因编辑方法的临床前评估

线粒体神经胃肠道脑肌病（MNGIE）是一种由编码胸苷磷酸化酶（TP）的TYMP基因隐性突变引起的罕见疾病。在这项研究中，通过 CRISPR/Cas9 和TYMP cDNA的协同递送和活性，实现了TYMP转基因有效整合到 MNGIE小鼠模型中肝细胞Tymp和Alb基因座的内含子中。CRISPR/Cas9 分别使用脂质纳米颗粒 (LNP) 或聚合物纳米颗粒作为 mRNA 递送，或在 AAV2/8 病毒载体中递送；后者也用于包装TYMP cDNA。在Tymp下游插入 cDNA 模板和Alb启动子确保转基因表达。使用携带 CRISPR/Cas9 mRNA 的 LNP 获得了最好的体内结果。接受治疗的小鼠血浆核苷（胸苷和脱氧尿苷）水平长期（1 年）降低，这与肝细胞中TYMP mRNA 和功能性酶的存在相关。在具有编辑的Alb基因座的小鼠中，转基因产生了一种分泌的杂合 Alb-hTP 蛋白，在血浆中检测到超生理水平的 TP 活性。在Tymp编辑的小鼠中获得了相同的结果轨迹。最后，在没有核酸酶的情况下，在仅用含有 DNA 模板的 AAV 载体处理的动物中发现了某种程度的基因编辑，尽管对血浆核苷水平没有影响。总体而言，这些结果证明了肝脏定向基因组编辑在 MNGIE 长期校正中的可行性，与其他方法相比具有几个优势。