Efficient and safe therapeutic use of paired Cas9-nickases for primary hyperoxaluria type 1

Abstract

The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. In addition, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.

Recent Therapeutic Gene Editing Applications to Genetic Disorders

“…However, off-target indels were not systematically evaluated. In a different approach to minimizing off-target effects, a paired-D10ASaCas9n strategy was delivered with the same two sgRNAs by two AAV8 to disrupt Hao1 in the same mouse model [139]. The authors reported 57% editing efficiency in Hao1 (Table 4) and a lower AAV integration rate compared to Cas9 nuclease, resulting in reduced GO protein expression and decreased oxalate accumulation in PH1 mice.…”