Phenylalanine hydroxylase (PAH) deficiency, colloquially known as phenylketonuria (PKU), is among the most common inborn errors of metabolism and in the past decade has become a target for the development of novel therapeutics such as gene therapy. PAH deficient mouse models have been key to new treatment development, but all prior existing models natively express liver PAH polypeptide as inactive or partially active PAH monomers, which complicates the experimental assessment of protein expression following therapeutic gene, mRNA, protein, or cell transfer. The mutant PAH monomers are able to form hetero-tetramers with and inhibit the overall holoenzyme activity of wild type PAH monomers produced from a therapeutic vector. Preclinical therapeutic studies would benefit from a PKU model that completely lacks both PAH activity and protein expression in liver. In this study, we employed CRISPR/Cas9-mediated gene editing in fertilized mouse embryos to generate a novel mouse model that lacks exon 1 of the Pah gene. Mice that are homozygous for the Pah exon 1 deletion are viable, severely hyperphenylalaninemic, accurately replicate phenotypic features of untreated human classical PKU and lack any detectable liver PAH activity or protein. This model of classical PKU is ideal for further development of gene and cell biologics to treat PKU.

苯丙氨酸羟化酶（PAH）缺乏症，俗称苯丙酮尿症（PKU），是最常见的先天性代谢错误，在过去的十年中，它已成为开发新疗法（如基因疗法）的目标。PAH缺陷小鼠模型一直是新疗法发展的关键，但是所有现有模型都天然将肝PAH多肽表达为无活性或部分活性的PAH单体，这使得在治疗性基因，mRNA，蛋白质或细胞转移后蛋白质表达的实验评估变得复杂。突变的PAH单体能够与由治疗载体产生的野生型PAH单体形成异四聚体并抑制其整体全酶活性。临床前治疗研究将受益于完全缺乏PAH活性和肝脏蛋白质表达的PKU模型。在这项研究中，我们在受精的小鼠胚胎中使用了CRISPR / Cas9介导的基因编辑功能，以生成一个缺少小鼠第1外显子的新型小鼠模型。Pah基因。Pah外显子1缺失纯合子的小鼠是可行的，严重的高苯丙氨酸血症，可准确复制未经治疗的人类经典PKU的表型特征，并且缺乏任何可检测到的肝PAH活性或蛋白质。这种经典的PKU模型非常适合进一步开发基因和细胞生物制剂来治疗PKU。