Knockdown of lactate dehydrogenase by adeno-associated virus-delivered CRISPR/Cas9 system alleviates primary hyperoxaluria type 1.
Knockdown of lactate dehydrogenase by adeno-associated virus-delivered CRISPR/Cas9 system alleviates primary hyperoxaluria type 1.
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DOI：
10.1002/ctm2.261
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发表时间：
2020-12
影响因子：
10.6
通讯作者：
Geng H
中科院分区：
文献类型：
--
作者：
Zheng R;Fang X;Chen X;Huang Y;Xu G;He L;Li Y;Niu X;Yang L;Wang L;Li D;Geng H
原发性高草酸尿1型(PH1)是一种罕见的遗传性疾病，由肝脏草酸内源性过多引起，导致高草酸尿、草酸钙肾结石复发和终末期肾病。乳酸脱氢酶(LDH)是减少草酸产生的理想靶点，因为它负责肝脏中乙醛到草酸的转化，草酸代谢的最后一步。在这里，我们研究了聚集性规则间隔短回文重复序列(CRISPR)/Cas9技术通过特异性干扰肝脏LDH来改善PH1的疗效和潜在副作用。用嗜铬细胞瘤(PC12)细胞在体外评价单引导RNA的切割效果。PH1新生大鼠被单次注射腺相关病毒来传递靶向LDH的CRISPR/Cas9系统。注射三周后，进行肝脏活检，以检测LDH表达、肝脏损伤和肝脏代谢组学。定期监测尿草酸，并在0.5%乙二醇组4周后评估肾脏草酸钙沉积。治疗6个月后，对动物实施安乐死，取肝外器官进行毒性分析。治疗组PH1大鼠肝细胞中有20%的LDHA基因被特异性敲除，导致LDH的表达比对照组低50%。与对照组相比，在整个6个月的研究期间，治疗组的尿草酸水平显著降低，肾脏草酸钙沉淀显著减轻。虽然没有检测到CRISPR/Cas9相关的非靶标编辑或肝脏毒性，但我们观察到肝脏三羧酸(TCA)和糖酵解途径的轻微代谢变化。CRISPR/Cas9介导的LDH中断可能代表了一种适用的新策略，以缓解PH1的长期影响和低编辑效率要求。CRISPR/Cas9技术通过靶向非必需的酶为遗传单基因疾病提供了新的方法。利用大鼠模型，我们证明了AAV递送的CRISPR/Cas9系统是一个有效和安全的平台，可以下调乳酸脱氢酶并缓解威胁生命的原发性高草酸尿症1型。
Primary hyperoxaluria type 1 (PH1) is a rare genetic disorder caused by endogenous overproduction of hepatic oxalate, leading to hyperoxaluria, recurrent calcium oxalate kidney stones, and end‐stage renal disease. Lactate dehydrogenase (LDH) is an ideal target for diminishing oxalate production as it is responsible for glyoxylate to oxalate conversion in the liver, the last step of oxalate metabolism. Here, we investigated the therapeutic efficacy and potential side effects of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to ameliorate PH1 via specifically disrupting the hepatic LDH. Pheochromocytoma (PC12) cells were used to assess the efficacy of cleavage of single‐guide RNAs in vitro. PH1 neonatal rats were injected with a single administration of adeno‐associated virus to deliver the CRISPR/Cas9 system that targeted LDH. Three weeks after injection, a liver biopsy was performed to detect LDH expression, liver injury, and liver metabolomics. Urinary oxalate was regularly monitored, and renal calcium oxalate deposition was evaluated after 4 weeks of 0.5% ethylene glycol challenge. After 6 months of treatment, animals were euthanized, and ex‐liver organs were harvested for toxicity analysis. The Ldha gene was specifically knocked out in 20% of the liver cells of PH1 rats in the treatment group, leading to a 50% lower LDH expression than that in the control group. Compared to the control groups, urinary oxalate levels were significantly decreased, and renal calcium oxalate precipitation was largely mitigated in the treatment group throughout the entire 6‐month study period. While no CRISPR/Cas9‐associated off‐target edits or hepatotoxicity were detected, we observed mild metabolic changes in the liver tricarboxylic acid (TCA) and glycolysis pathways. CRISPR/Cas9‐mediated LDH disruption may represent an applicable new strategy for alleviating PH1 for its long‐lasting effect and low editorial efficiency requirements. CRISPR/Cas9 technology provides novel approaches for inherited monogenic diseases by targeting non‐essential enzymes. Using a rat model, we demonstrate that the AAV‐delivered CRISPR/Cas9 system is an effective and safe platform for knocking down lactate dehydrogenase and alleviating the life‐threatening primary hyperoxaluria type 1.
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