Allele-specific antisense oligonucleotide treatment rescues atad3-associated phenotype in zebrafish.

Pathogenic variants in ATAD3A cause a spectrum of multisystem disorders, with a recurrent dominant-negative variant (c.1582C>T; p.Arg528Trp) associated with neurodevelopmental disease. Given the tolerance of ATAD3A to heterozygous loss of function variants, allele-specific transcript reduction represents a promising therapeutic strategy. We designed and optimized allele-specific antisense oligonucleotides (ASOs) targeting the c.1582C>T transcript and evaluated their efficacy and specificity in affected fibroblasts using allele-specific primers and amplicon-based next generation sequencing. Therapeutic potential was further assessed in vivo in zebrafish embryos expressing human wild-type or mutant ATAD3A transcripts. An optimized gapmer ASO selectively reduced mutant ATAD3A transcripts while relatively sparing the wild-type allele. In addition to RNase H-mediated degradation, the ASO induced exon skipping, leading to degradation of the aberrant transcript without production of a truncated protein. In zebrafish, expression of mutant human ATAD3A in embryos caused developmental abnormalities including reduced eye size, which were robustly rescued by co-injection of the optimized ASO. Our findings provide proof of concept for allele-targeted ASO therapy for dominant-negative ATAD3A variants. This work highlights the therapeutic potential of ASOs for rare dominant disorders involving genes tolerant to heterozygous loss-of-function, and establishes zebrafish as a versatile platform for in vivo ASO optimization.