C1 - Base Editing and Prime Editing
679: Systematic Off-Target Analyses of Base-Editing Therapeutic Leads Show Reduced Off-Target Editing, Reduced Bystander Editing, and Increased On-Target Editing by Hybrid Guide RNAs
Type: Poster Session
Poster Board Number: 679
Presentation Details
Session Title: Wednesday Posters: Base Editing and Prime Editing
Phenylketonuria (PKU) and hereditary tyrosinemia type 1 (HT1) are autosomal recessive disorders linked to the phenylalanine hydroxylase (PAH) gene and fumarylacetoacetate hydrolase (FAH) gene, respectively. Potential therapeutic strategies to ameliorate disease include corrective editing of pathogenic variants in the PAH gene 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 recurrent PAH variants 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 with OligoNucleotide Enrichment and sequencing (ONE-seq) identified certain hybrid gRNAs that dramatically reduced off-target editing and reduced bystander editing, while actually increasing on-target editing either in vitro or in vivo, e.g., in a humanized PKU mouse model treated with lipid nanoparticles (LNPs) with adenine base editor mRNA and a standard versus hybrid gRNA. These studies highlight the remarkable utility of hybrid gRNAs to improve the safety and efficacy of base-editing therapies. Moreover, they recommend a novel strategy for de-risking of LNP editing therapies: rather than the usual approach of assessing a standard gRNA by techniques such as ONE-seq, compiling a large list of nominated candidate genomic sites, and extensively analyzing the candidate sites for off-target editing in treated cells—instead using variant-aware ONE-seq to screen hybrid gRNAs and identify a hybrid gRNA that has preserved or improved on-target editing while reducing the number of nominated candidate off-target sites requiring further evaluation to a few or even zero.
Dominique L. Brooks1, Madelynn Whittaker1, Aidan Quigley1, Ishaan Jindal1, Rebecca Ahrens-Nicklas2, Kiran Musunuru1, William Peranteau2, Xiao Wang1
1Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA,2Children's Hospital of Philadelphia, Philadelphia, PA"
Find This Session