Targeting key enzymes that generate oxalate precursors or substrates is an alternative strategy to eliminate primary hyperoxaluria type I(PH1),the most common and lifethreatening type of primary hyperoxaluria.The comp...Targeting key enzymes that generate oxalate precursors or substrates is an alternative strategy to eliminate primary hyperoxaluria type I(PH1),the most common and lifethreatening 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 Cpf1system 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 Hao1and 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 offtarget effects were detected.We demonstrated the AAVAsCpf1 system can target multiple genes and rescue the pathogenic phenotype in PH1,serving as a proof-ofconcept for the development of multiplex genome editingbased gene therapy.展开更多
Primary hyperoxaluria type 1(PH1)is a severe hereditary disease,leading to the accumulation of oxalate in multiple organs,particularly the kidney.Hydroxyacid oxidase 1(HAO1),a pivotal gene involved in oxalate producti...Primary hyperoxaluria type 1(PH1)is a severe hereditary disease,leading to the accumulation of oxalate in multiple organs,particularly the kidney.Hydroxyacid oxidase 1(HAO1),a pivotal gene involved in oxalate production,is an approved target for the treatment of PH1.In this study,we demonstrated the discovery of several novel therapeutic sites of the Hao1 gene and the efficient editing of Hao1c.290-2 A in vivo with lipid nanoparticles(LNP)delivered adenine base editing(ABE)mRNA.A single infusion of LNP-ABE resulted in a near-complete knockout of Hao1 in the liver,leading to the sustainable normalization of urinary oxalate(for at least 6 months)and complete rescue of the pathophysiology in PH1 rats.Additionally,a significant correlation between Hao1 editing efficiency and urinary oxalate levels was observed and over 60%Hao1 editing efficiency was required to achieve the normalization of urinary oxalate in PH1 rats.These findings suggest that the LNP-mediated base-editing of Hao1c.290-2 A is an efficient and safe approach to PH1 therapy,highlighting its potential utility in clinical settings.展开更多
基金partially supported by the Science and Technology Commission of Shanghai Municipality (22YF1426900,20140900200)National Natural Science Foundation of China (32001057)。
文摘Targeting key enzymes that generate oxalate precursors or substrates is an alternative strategy to eliminate primary hyperoxaluria type I(PH1),the most common and lifethreatening 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 Cpf1system 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 Hao1and 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 offtarget effects were detected.We demonstrated the AAVAsCpf1 system can target multiple genes and rescue the pathogenic phenotype in PH1,serving as a proof-ofconcept for the development of multiplex genome editingbased gene therapy.
基金partially supported by the National Natural Science Foundation of China (82470794,32025023 and 32230064)the National Key Research and Development Program of China (2023YFC3403400 to Li,D)a grant from the Science and Technology Commission of Shanghai Municipality (22YF1426900)。
文摘Primary hyperoxaluria type 1(PH1)is a severe hereditary disease,leading to the accumulation of oxalate in multiple organs,particularly the kidney.Hydroxyacid oxidase 1(HAO1),a pivotal gene involved in oxalate production,is an approved target for the treatment of PH1.In this study,we demonstrated the discovery of several novel therapeutic sites of the Hao1 gene and the efficient editing of Hao1c.290-2 A in vivo with lipid nanoparticles(LNP)delivered adenine base editing(ABE)mRNA.A single infusion of LNP-ABE resulted in a near-complete knockout of Hao1 in the liver,leading to the sustainable normalization of urinary oxalate(for at least 6 months)and complete rescue of the pathophysiology in PH1 rats.Additionally,a significant correlation between Hao1 editing efficiency and urinary oxalate levels was observed and over 60%Hao1 editing efficiency was required to achieve the normalization of urinary oxalate in PH1 rats.These findings suggest that the LNP-mediated base-editing of Hao1c.290-2 A is an efficient and safe approach to PH1 therapy,highlighting its potential utility in clinical settings.
