A mutation-independent AAV-based gene therapy approach to treat Phenylketonuria

Abstract

Phenylketonuria (PKU) is an autosomal recessive disorder of phenylalanine metabolism, primarily caused by mutations in the phenylalanine hydroxylase (PAH) gene, resulting in the toxic accumulation of phenylalanine. Affected infants are born healthy, but without intervention, can rapidly experience cognitive and developmental delays. Although newborn screening and early dietary intervention prevent neurological damage, long-term dietary adherence is burdensome, and existing pharmacological interventions benefit only a subset of patients. Consequently, there is a real need for broadly applicable, long-term treatment options. Gene therapy holds promise for addressing this unmet need. This thesis aimed to introduce a functional copy of human PAH cDNA into the first intron of the endogenous Pah locus in a murine PKU model using a homology-independent targeted integration (HITI) approach. A dual AAV CRISPR/SaCas9 and HITI donor strategy incorporating a 2A element was developed to enable bicistronic expression under endogenous promoter control, providing a mutation-agnostic approach capable of treating all PKU variants. Proof-of-concept studies with GFP donor demonstrated efficient genomic targeting, robust expression in up to 70% of hepatocytes, and production of hybrid transcripts, confirming on-target integration. Therapeutic PAH donors exhibited similar integration and expression, although significant reductions in blood phenylalanine levels were not observed. These findings highlight both the promise and the challenges of gene editing in treating PKU. While targeted integration at the Pah locus is feasible, translation into meaningful metabolic correction will require improvements in nuclease and donor delivery. Emerging dual-modality approaches, such as using lipid nanoparticle-mediated Cas9 delivery with AAV-donor, may enhance safety and efficiency. Overall, this work supports the potential of mutation-agnostic genome editing for treating PKU.