R2Tg retrotransposon mediated targeted integration of exogenous genes into the 28S rDNA locus in mammalian cell lines for PKU gene therapy.

Abstract

Phenylketonuria (PKU) remains a challenging genetic disorder due to the absence of therapeutic strategies capable of achieving sustained restoration of phenylalanine hydroxylase (PAH) function. Here, we employed an R2 retrotransposon in Taeniopygia guttata (R2Tg)-based system to enable targeted integration of exogenous PAH mRNA into the 28S ribosomal DNA (rDNA) locus of the host genome, with the goal of rescuing PAH deficiency caused by loss-of-function mutations. Experimental results demonstrated that lipid nanoparticle (LNP) delivery of R2Tg facilitated efficient site-specific integration of EGFP and PAH transgenes in mammalian cells, significantly elevating PAH mRNA and protein expression without disrupting endogenous 28S rRNA levels. These findings support the potential of this strategy as a precise genomic modification tool with promise for lifelong correction of PAH deficiency.