Targeting key enzymes that generate oxalate precursors or substrates is an alternative strategy to eliminate primary hyperoxaluria type I (PH1), the most common and life-threatening type of primary hyperoxaluria. The compact Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) from the Prevotella and Francisella 1 (Cpf1) protein simplifies multiplex gene editing and allows for all-in-one adeno-associated virus (AAV) delivery. We hypothesized that the multiplex capabilities of the Cpf1 system could help minimize oxalate formation in PH1 by simultaneously targeting the hepatic hydroxyacid oxidase 1 ( Hao1) and lactate dehydrogenase A ( Ldha) genes. Study cohorts included treated PH1 rats ( Agxt Q84X rats injected with AAV-AsCpf1 at 7 days of age), phosphate-buffered saline (PBS)-injected PH1 rats, untreated PH1 rats, and age-matched wild-type (WT) rats. The most efficient and specific CRISPR RNA (crRNA) pairs targeting the rat Hao1 and Ldha genes were initially screened ex vivo. In vivo experiments demonstrated efficient genome editing of the Hao1 and Ldha genes, primarily resulting in small deletions. This resulted in decreased transcription and translational expression of Hao1 and Ldha. Treatment significantly reduced urine oxalate levels, reduced kidney damage, and alleviated nephrocalcinosis in rats with PH1. No liver toxicity, ex-liver genome editing, or obvious off-target effects were detected. We demonstrated the AAV-AsCpf1 system can target multiple genes and rescue the pathogenic phenotype in PH1, serving as a proof-of-concept for the development of multiplex genome editing-based gene therapy.
1型原发性高草酸尿症（PH1）是一种罕见的代谢性疾病，由 AGXT突变引起肝脏草酸大量产生，继而导致尿路结石和终末期肾病等严重后果，危及生命。此前只能通过肝脏-肾脏联合移植根治疾病。目前最有前景的治疗策略是通过阻断草酸合成上游的关键酶，如 HAO1和 LDHA基因编码酶，从根源上减少肝脏草酸合成。相比传统CRISPR/Cas9，Cpf1基因编辑工具拥有单个crRNA执行多重基因编辑、更低的脱靶效应、体积更小等优势，可以通过单个腺相关病毒（AAV）进行包装。基于研究团队前期报道的PH1大鼠模型，该研究首次尝试体内多基因编辑，使用Cpf1系统同时靶向PH1大鼠肝脏中的 Hao1和 Ldha基因。实验结果表明，AAV-Cpf1系统可以同时高效地编辑这两个基因，相应的GO和LDH蛋白表达显著降低。疗效上，双基因编辑使得PH1大鼠的尿草酸水平显著降低，并减轻了肾脏损伤和肾草酸钙结晶。安全性上，该方案没有产生肝损伤，亦无检测到基因组脱靶编辑与肝外器官的非特异性编辑。这一研究成果为PH1等代谢性疾病的治疗提供了新思路，为基于多重基因组编辑的基因疗法开发提供了经验参考。.
Keywords: Genome editing; Hydroxyacid oxidase 1; Hyperoxaluria; Lactate dehydrogenase.