Department of Pediatrics, Oregon Stem Cell Center, Oregon Health and Science University, Portland, Oregon, USA.
Department of Pathology, Texas Children's Hospital, Houston, Texas, USA.
Hum Gene Ther. 2021 Mar;32(5-6):294-301. doi: 10.1089/hum.2020.042. Epub 2020 Oct 16.
The efficiency of gene repair by homologous recombination in the liver is enhanced by CRISP/Cas9 incision near the mutation. In this study, we explored interventions designed to further enhance hepatocyte gene repair in a model of hereditary tyrosinemia. A two-AAV system was employed: one virus carried a Staphylococcus pyogenes Cas9 (SpCas9) expression cassette and the other harbored a U6 promoter-driven sgRNA and a fragment of fumarylacetoacetate hydrolase () genomic DNA as the homologous recombination donor. In neonatal mice, a gene correction frequency of ∼10.8% of hepatocytes was achieved. The efficiency in adult mice was significantly lower at ∼1.6%. To determine whether hepatocyte replication could enhance the targeting frequency, cell division was induced with thyroid hormone T3. This more than doubled the gene correction efficiency to 3.5% ( < 0.005). To determine whether SpCas9 delivery was rate limiting, the gene repair AAV was administered to SpCas9 transgenic mice. However, this did not significantly enhance gene repair. Finally, we tested whether the Fanconi anemia (FA) DNA repair pathway was important in hepatocyte gene repair. Gene correction frequencies were significantly lower in neonatal mice lacking the FA complementation group A () gene. Taken together, we conclude that pharmacological induction of hepatocyte replication along with manipulation of DNA repair pathways could be a useful strategy for enhancing gene correction.
同源重组修复基因的效率通过 CRISPR/Cas9 在突变附近的切割得到增强。在这项研究中，我们探索了旨在进一步增强遗传性酪氨酸血症模型中肝细胞基因修复的干预措施。采用了双 AAV 系统：一种病毒携带酿脓链球菌 Cas9（SpCas9）表达盒，另一种携带 U6 启动子驱动的 sgRNA 和延胡索酰乙酰乙酸水解酶（）基因组 DNA 片段作为同源重组供体。在新生小鼠中，实现了约 10.8%的肝细胞基因校正频率。在成年小鼠中的效率明显较低，约为 1.6%。为了确定肝细胞复制是否可以增强靶向频率，用甲状腺激素 T3 诱导细胞分裂。这将基因校正效率提高了两倍多，达到 3.5%（<0.005）。为了确定 SpCas9 递呈是否是限速步骤，将基因修复 AAV 递呈给 SpCas9 转基因小鼠。然而，这并没有显著增强基因修复。最后，我们测试了范可尼贫血（FA）DNA 修复途径在肝细胞基因修复中的重要性。缺乏 FA 补体组 A（）基因的新生小鼠的基因校正频率明显降低。综上所述，我们得出结论，药理学诱导肝细胞复制并操纵 DNA 修复途径可能是增强基因校正的有效策略。
