Phenylketonuria (PKU) due to recessively inherited phenylalanine hydroxylase (PAH) deficiency results in hyperphenylalaninemia, which is toxic to the central nervous system. Restriction of dietary phenylalanine intake remains the standard of PKU care and prevents the major neurologic manifestations of the disease, yet shortcomings of dietary therapy remain, including poor adherence to a difficult and unpalatable diet, an increased incidence of neuropsychiatric illness, and imperfect neurocognitive outcomes. Gene therapy for PKU is a promising novel approach to promote lifelong neurological protection while allowing unrestricted dietary phenylalanine intake. In this study, liver-tropic recombinant AAV2/8 vectors were used to deliver CRISPR/Cas9 machinery and facilitate correction of the Pah enu2 allele by homologous recombination. Additionally, a non-homologous end joining (NHEJ) inhibitor, vanillin, was co-administered with the viral drug to promote homology-directed repair (HDR) with the AAV-provided repair template. This combinatorial drug administration allowed for lifelong, permanent correction of the Pah enu2 allele in a portion of treated hepatocytes of mice with PKU, yielding partial restoration of liver PAH activity, substantial reduction of blood phenylalanine, and prevention of maternal PKU effects during breeding. This work reveals that CRISPR/Cas9 gene editing is a promising tool for permanent PKU gene editing.

中文翻译：

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AAV介导的小鼠苯丙酮尿症的CRISPR / Cas9基因编辑。

由于隐性遗传的苯丙氨酸羟化酶（PAH）缺乏而引起的苯丙酮尿​​症（PKU）会导致高苯丙氨酸血症，对中枢神经系统有毒。限制饮食中苯丙氨酸的摄入仍然是北大联盟护理的标准，可以预防该疾病的主要神经系统表现，但是饮食疗法的缺点仍然存在，包括依从困难和不愉快的饮食的依从性差，神经精神疾病的发病率增加以及神经认知结果不完善。PKU的基因疗法是一种有前途的新颖方法，可促进终身神经保护，同时允许饮食中苯丙氨酸的摄入不受限制。在这项研究中，嗜肝重组AAV2 / 8载体被用于提供CRISPR / Cas9机制，并通过同源重组促进Pah enu2等位基因的校正。此外，将非同源末端连接（NHEJ）抑制剂香草醛与病毒药物共同给药，以通过AAV提供的修复模板促进同源性定向修复（HDR）。这种组合药物的使用可以终身，永久性纠正PKU小鼠肝细胞的一部分中的Pah enu2等位基因，从而部分恢复肝脏PAH活性，显着降低血液苯丙氨酸的含量，并防止母体PKU在繁殖过程中的影响。这项工作表明，CRISPR / Cas9基因编辑是用于永久性PKU基因编辑的有前途的工具。永久性纠正PKU小鼠经治疗的部分肝细胞中Pah enu2等位基因，可部分恢复肝脏PAH活性，显着降低血液中的苯丙氨酸含量，并防止母体PKU繁殖。这项工作表明，CRISPR / Cas9基因编辑是用于永久性PKU基因编辑的有前途的工具。永久性纠正PKU小鼠经治疗的部分肝细胞中Pah enu2等位基因，可部分恢复肝脏PAH活性，显着降低血液中的苯丙氨酸含量，并防止母体PKU繁殖。这项工作表明，CRISPR / Cas9基因编辑是用于永久性PKU基因编辑的有前途的工具。