Rapid, definitive treatment of phenylketonuria in variant-humanized mice with corrective editing

Phenylketonuria (PKU), an autosomal recessive disorder caused by pathogenic variants in the phenylalanine hydroxylase (PAH) gene, results in the accumulation of blood phenylalanine (Phe) to neurotoxic levels. Current dietary and medical treatments are chronic and reduce, rather than normalize, blood Phe levels. Among the most frequently occurring PAH variants in PKU patients is the P281L (c.842C>T) variant. Using a CRISPR prime-edited hepatocyte cell line and a humanized PKU mouse model, we demonstrate efficient in vitro and in vivo correction of the P281L variant with adenine base editing. With delivery of ABE8.8 mRNA and either of two guide RNAs in vivo using lipid nanoparticles (LNPs) in humanized PKU mice, we observe complete and durable normalization of blood Phe levels within 48 hours of treatment, resulting from corrective PAH editing in the liver. These studies nominate a drug candidate for further development as a definitive treatment for a subset of PKU patients. All the next-generation sequencing data from cells are uploaded here.

苯丙酮尿症（Phenylketonuria, PKU）是一种由苯丙氨酸羟化酶（phenylalanine hydroxylase, PAH）基因致病变异引发的常染色体隐性遗传病，可导致血液中苯丙氨酸（phenylalanine, Phe）蓄积至神经毒性水平。当前的饮食与药物治疗均为长期干预手段，仅能降低而非使血Phe水平恢复至正常范围。在PKU患者中，最为常见的PAH致病变异之一为P281L（c.842C>T）变异。本研究借助CRISPR先导编辑的肝细胞系与人源化PKU小鼠模型，证实腺嘌呤碱基编辑可在体外与体内高效校正P281L变异。通过脂质纳米颗粒（lipid nanoparticles, LNPs）将ABE8.8 mRNA与两种向导RNA之一递送至人源化PKU小鼠体内后，我们观察到给药后48小时内血液Phe水平即实现完全且持久的正常化，该效果源于肝脏内PAH基因的校正性编辑。本研究为针对部分PKU患者的根治性治疗筛选出了候选药物，可用于后续开发。本研究中所有来自细胞的下一代测序数据已上传至本数据集。