DNA & RNA Medicine Division, Center for Applied Medical Research (CIMA), University of Navarra, 31008, Pamplona, Spain.
Institute for Transfusion Medicine and Gene Therapy, Medical Center - University of Freiburg, 79106, Freiburg, Germany.
EMBO Mol Med. 2024 Jan;16(1):112-131. doi: 10.1038/s44321-023-00008-8. Epub 2024 Jan 5.
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.
腺相关病毒载体（AAV）介导的 CRISPR-Cas9 基因敲除治疗的应用受到潜在的脱靶修饰和载体基因组失控整合到 CRISPR 介导的双链断裂的风险的限制。为了解决这些问题，我们探索了使用 AAV 传递的成对金黄色葡萄球菌核酸酶（D10ASaCas9）来靶向 Hao1 基因，以治疗 1 型原发性高草酸尿症（PH1）。我们的研究表明，该方法可以有效敲除 Hao1 基因，显著降低乙二醇氧化酶的表达，并在 PH1 小鼠中产生治疗效果。通过 CIRCLE-seq 和 CAST-Seq 分析评估非预期的遗传修饰，未发现脱靶活性或染色体易位。重要的是，与 SaCas9 核酸酶相比，使用成对 D10ASaCas9 可显著减少目标位点的 AAV 整合。此外，我们的研究强调了当前分析工具在表征成对 D10ASaCas9 引入的修饰方面的局限性，需要开发定制的管道以进行更准确的表征。这些结果描述了朝着为 PH1 患者提供安全有效的潜在长期治疗方法的积极进展。
