Adenine base editing corrects point mutation in mitochondrial single-stranded binding protein (SSBP1) to improve mitochondrial function

Mutations in nuclear genes that regulate the mitochondrial DNA (mtDNA) replication machinery have been linked to mtDNA depletion syndromes. Through whole-genome sequencing, we identified a heterozygous missense mutation (c.272G>A:p.Arg91Gln) in single-stranded binding protein 1 (SSBP1), a crucial protein involved in mtDNA replication. The proband manifested symptoms including sensorineural deafness, congenital cataract, optic atrophy, macular dystrophy, and myopathy, all of which are compatible with mtDNA depletion disease. We found that this SSBP1 mutation impeded multimer formation and DNA-binding affinity, which led to reduced efficiency of mtDNA replication and altered mitochondria dynamics. To correct this mutation, we tested two adenine base editor (ABE) variants using patient-derived fibroblasts. One variant, NG-Cas9-based ABE8e (NG-ABE8e), exhibited higher editing efficacy (up to 30% editing), and edited cells showed signs of improved mitochondrial replication and function, including increased mtDNA and ATP production. However, these cells also had higher frequencies of off-target editing of nearby nucleotides, but the risks from bystander editing were limited due to mostly silent mutations and off-target sites in non-translated regions. The other variant, NG-Cas9-based ABE8eWQ (NG-ABE8eWQ), had a safer therapeutic profile with very few off-target effects, but this came at the cost of lower editing efficacy (up to 10% editing). Despite this, NG-ABE8eWQ-edited cells still restored replication and improved mtDNA copy number, which in turn recovery of compromised mitochondrial function. As these therapeutic strategies make their way into the clinic, our research suggests that base editing-based gene therapies may be a promising treatment for mitochondrial diseases, including those associated with SSBP1 mutations.