Search

Phenylketonuria (PKU), hereditary tyrosinemia type 1 (HT1), and mucopolysaccharidosis type 1 (MPSI) are autosomal recessive disorders linked to the phenylalanine hydroxylase ( PAH ) gene, fumarylacetoacetate hydrolase ( FAH ) gene, and alpha-L-iduronidase ( IDUA ) gene, respectively. Potential therapeutic strategies to ameliorate disease include corrective editing of pathogenic variants in the PAH and IDUA genes and, as a variant-agnostic approach, inactivation of the 4-hydroxyphenylpyruvate dioxygenase ( HPD ) gene, a modifier of HT1, via adenine base editing. Here we evaluated the off-target editing profiles of therapeutic lead guide RNAs (gRNAs) that, when combined with adenine base editors correct the recurrent PAH P281L variant, PAH R408W variant, or IDUA W402X variant or disrupt the HPD gene in human hepatocytes. To mitigate off-target mutagenesis, we systematically screened hybrid gRNAs with DNA nucleotide substitutions. Comprehensive and variant-aware specificity profiling of these hybrid gRNAs reveal dramatically reduced off-target editing and reduced bystander editing. Lastly, in a humanized PAH P281L mouse model, we showed that when formulated in lipid nanoparticles (LNPs) with adenine base editor mRNA, selected hybrid gRNAs revert the PKU phenotype, substantially enhance on-target editing, and reduce bystander editing in vivo . These studies highlight the utility of hybrid gRNAs to improve the safety and efficacy of base-editing therapies., Competing Interests: M.N.W. is a fellow with Artis Ventures. J.Z.W. is an employee of Agilent Technologies. R.C.A.-N. is an advisor to Latus Bio. K.M. is an advisor to and holds equity in Verve Therapeutics and Variant Bio, is an advisor to LEXEO Therapeutics, and receives research funding from Nava Therapeutics and Beam Therapeutics. M.-G.A. is an advisor to Afrigen Biologics. The University of Pennsylvania and Children’s Hospital of Philadelphia have filed patent applications related to the use of base editing for the treatment of phenylketonuria and tyrosinemia (inventors include M.N.W., D.L.B., R.C.A.-N., K.M., W.H.P., and X.W.).The remaining authors declare no competing interests.