Knockdown of lactate dehydrogenase by adeno‐associated virus‐delivered CRISPR/Cas9 system alleviates primary hyperoxaluria type 1
Abstract Background 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 oxa...
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2020-12-01
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Series: | Clinical and Translational Medicine |
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Online Access: | https://doi.org/10.1002/ctm2.261 |
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doaj-218597d517ff46b3b8b4ceebfb8ededb |
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Article |
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DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Rui Zheng Xiaoliang Fang Xi Chen Yunteng Huang Guofeng Xu Lei He Yueyan Li Xuran Niu Lei Yang Liren Wang Dali Li Hongquan Geng |
spellingShingle |
Rui Zheng Xiaoliang Fang Xi Chen Yunteng Huang Guofeng Xu Lei He Yueyan Li Xuran Niu Lei Yang Liren Wang Dali Li Hongquan Geng Knockdown of lactate dehydrogenase by adeno‐associated virus‐delivered CRISPR/Cas9 system alleviates primary hyperoxaluria type 1 Clinical and Translational Medicine adeno‐associated virus CRISPR/Cas9 gene therapy lactate dehydrogenase primary hyperoxaluria type 1 |
author_facet |
Rui Zheng Xiaoliang Fang Xi Chen Yunteng Huang Guofeng Xu Lei He Yueyan Li Xuran Niu Lei Yang Liren Wang Dali Li Hongquan Geng |
author_sort |
Rui Zheng |
title |
Knockdown of lactate dehydrogenase by adeno‐associated virus‐delivered CRISPR/Cas9 system alleviates primary hyperoxaluria type 1 |
title_short |
Knockdown of lactate dehydrogenase by adeno‐associated virus‐delivered CRISPR/Cas9 system alleviates primary hyperoxaluria type 1 |
title_full |
Knockdown of lactate dehydrogenase by adeno‐associated virus‐delivered CRISPR/Cas9 system alleviates primary hyperoxaluria type 1 |
title_fullStr |
Knockdown of lactate dehydrogenase by adeno‐associated virus‐delivered CRISPR/Cas9 system alleviates primary hyperoxaluria type 1 |
title_full_unstemmed |
Knockdown of lactate dehydrogenase by adeno‐associated virus‐delivered CRISPR/Cas9 system alleviates primary hyperoxaluria type 1 |
title_sort |
knockdown of lactate dehydrogenase by adeno‐associated virus‐delivered crispr/cas9 system alleviates primary hyperoxaluria type 1 |
publisher |
Wiley |
series |
Clinical and Translational Medicine |
issn |
2001-1326 |
publishDate |
2020-12-01 |
description |
Abstract Background 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. Methods 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. Results 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. Conclusions CRISPR/Cas9‐mediated LDH disruption may represent an applicable new strategy for alleviating PH1 for its long‐lasting effect and low editorial efficiency requirements. |
topic |
adeno‐associated virus CRISPR/Cas9 gene therapy lactate dehydrogenase primary hyperoxaluria type 1 |
url |
https://doi.org/10.1002/ctm2.261 |
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doaj-218597d517ff46b3b8b4ceebfb8ededb2021-05-07T14:40:31ZengWileyClinical and Translational Medicine2001-13262020-12-01108n/an/a10.1002/ctm2.261Knockdown of lactate dehydrogenase by adeno‐associated virus‐delivered CRISPR/Cas9 system alleviates primary hyperoxaluria type 1Rui Zheng0Xiaoliang Fang1Xi Chen2Yunteng Huang3Guofeng Xu4Lei He5Yueyan Li6Xuran Niu7Lei Yang8Liren Wang9Dali Li10Hongquan Geng11Department of Pediatric Urology Xinhua Hospital, Shanghai Jiao Tong University School of Medicine Shanghai ChinaDepartment of Pediatric Urology Xinhua Hospital, Shanghai Jiao Tong University School of Medicine Shanghai ChinaShanghai Key Laboratory of Regulatory Biology Institute of Biomedical Sciences and School of Life Sciences East China Normal University Shanghai ChinaChildren's Stone Treatment Center, National Health and Family Planning Commission of the People's Republic of China Shanghai ChinaDepartment of Pediatric Urology Xinhua Hospital, Shanghai Jiao Tong University School of Medicine Shanghai ChinaDepartment of Pediatric Urology Xinhua Hospital, Shanghai Jiao Tong University School of Medicine Shanghai ChinaDepartment of Pediatric Urology Xinhua Hospital, Shanghai Jiao Tong University School of Medicine Shanghai ChinaShanghai Key Laboratory of Regulatory Biology Institute of Biomedical Sciences and School of Life Sciences East China Normal University Shanghai ChinaShanghai Key Laboratory of Regulatory Biology Institute of Biomedical Sciences and School of Life Sciences East China Normal University Shanghai ChinaShanghai Key Laboratory of Regulatory Biology Institute of Biomedical Sciences and School of Life Sciences East China Normal University Shanghai ChinaShanghai Key Laboratory of Regulatory Biology Institute of Biomedical Sciences and School of Life Sciences East China Normal University Shanghai ChinaDepartment of Pediatric Urology Xinhua Hospital, Shanghai Jiao Tong University School of Medicine Shanghai ChinaAbstract Background 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. Methods 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. Results 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. Conclusions CRISPR/Cas9‐mediated LDH disruption may represent an applicable new strategy for alleviating PH1 for its long‐lasting effect and low editorial efficiency requirements.https://doi.org/10.1002/ctm2.261adeno‐associated virusCRISPR/Cas9gene therapylactate dehydrogenaseprimary hyperoxaluria type 1 |