Improved specificity and efficacy of base-editing therapies with hybrid guide RNAs.

Phenylketonuria (PKU), hereditary tyrosinemia type 1 (HT1), and mucopolysaccharidosis type 1 (MPSI) are autosomal recessive disorders linked to the phenylalanine hydroxylase () gene, fumarylacetoacetate hydrolase () gene, and alpha-L-iduronidase () gene, respectively. Potential therapeutic strategies to ameliorate disease include corrective editing of pathogenic variants in the and genes and, as a variant-agnostic approach, inactivation of the 4-hydroxyphenylpyruvate dioxygenase () 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 P281L variant, R408W variant, or W402X variant or disrupt the 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 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 . These studies highlight the utility of hybrid gRNAs to improve the safety and efficacy of base-editing therapies.