Abstract 3104: Adenine Base Editing To Correct Pathogenic Variants Causing Pseudoxanthoma Elasticum

Abstract

Pseudoxanthoma elasticum (PXE) is a rare, autosomal recessive disorder caused by pathogenic variants in ABCC6 , resulting in systemic depletion of the anti-mineralization compound pyrophosphate. Pyrophosphate depletion drives ectopic calcification of elastic fibers within the skin, retina, and vasculature. Arterial calcification can lead to intermittent claudication of peripheral arteries and coronary artery disease. There are currently no targeted therapies for PXE. Genome editing may be a promising therapeutic option because ABCC6 is predominantly expressed in the liver, a site that is amenable to current genome editing delivery modalities. We sought to assess whether adenine base editing could efficiently correct one of the most frequent ABCC6 variants in PXE patients, c.3490C>T (p.R1164X) in human hepatocytes and a humanized mouse model. We used prime editing to generate a homozygous R1164X human hepatoma cell line. Treatment of R1164X cells with mRNA encoding the adenine base editor ABE8.8 and a synthetic guide RNA (gRNA) resulted in efficient correction of the variant. OligoNucleotide Enrichment and sequencing (ONE-seq) was used to nominate candidate off-target sites, which were evaluated by targeted amplicon sequencing of edited R1164X cells. To mitigate off-target editing, we generated 21 hybrid gRNAs in which various RNA nucleotides in the protospacer were replaced by DNA nucleotides. All hybrid gRNAs maintained on-target editing, and interestingly, several hybrid gRNAs improved on-target editing compared to the standard gRNA. All hybrid gRNAs improved off-target editing, with multiple hybrid gRNAs eliminating off-target editing at multiple off-target sites. To assess in vivo correction of the R1164X variant, we generated a homozygous R1164X humanized mouse model which displays depletion of plasma pyrophosphate. Studies to correct the variant in the humanized mouse model by lipid nanoparticle delivery of the lead candidate hybrid gRNA are underway. Efficient correction of the R1164X variant in the liver by adenine base editing and restoration of pyrophosphate would represent one of the first examples of using genome editing to improve the multisystemic manifestations of a heritable connective tissue disorder.