Background and aim Hepatic ischemia–reperfusion injury(IRI)is a significant challenge in liver transplantation,trauma,hypovolemic shock,and hepatectomy,with limited effective interventions available.This study aimed ...Background and aim Hepatic ischemia–reperfusion injury(IRI)is a significant challenge in liver transplantation,trauma,hypovolemic shock,and hepatectomy,with limited effective interventions available.This study aimed to investigate the role of leukocyte cell-derived chemotaxin 2(LECT2)in hepatic IRI and assess the therapeutic potential of Lect2-short hairpin RNA(shRNA)delivered through adeno-associated virus(AAV)vectors.Materials and methods This study analyzed human liver and serum samples from five patients undergoing the Pringle maneuver.Lect2-knockout and C57BL/6J mice were used.Hepatic IRI was induced by clamping the hepatic pedicle.Treatments included recombinant human LECT2(rLECT2)and AAV-Lect2-shRNA.LECT2 expression levels and serum biomarkers including alanine aminotransferase(ALT),aspartate aminotransferase(AST),creatinine,and blood urea nitrogen(BUN)were measured.Histological analysis of liver necrosis and quantitative reverse-transcription polymerase chain reaction were performed.Results Serum and liver LECT2 levels were elevated during hepatic IRI.Serum LECT2 protein and mRNA levels increased post reperfusion.Lect2-knockout mice had reduced weight loss;hepatic necrosis;and serum ALT,AST,creatinine,and BUN levels.rLECT2 treatment exacerbated weight loss,hepatic necrosis,and serum biomarkers(ALT,AST,creatinine,and BUN).AAV-Lect2-shRNA treatment significantly reduced weight loss,hepatic necrosis,and serum biomarkers(ALT,AST,creatinine,and BUN),indicating therapeutic potential.Conclusions Elevated LECT2 levels during hepatic IRI increased liver damage.Genetic knockout or shRNA-mediated knockdown of Lect2 reduced liver damage,indicating its therapeutic potential.AAV-mediated Lect2-shRNA delivery mitigated hepatic IRI,offering a potential new treatment strategy to enhance clinical outcomes for patients undergoing liver-related surgeries or trauma.展开更多
常用的基因治疗表达载体有病毒表达载体和质粒表达载体,这两类传统基因治疗载体含有大量病毒或细菌DNA序列,会引发人体较严重的免疫反应、细胞炎症、细胞毒性副作用、以及基因表达沉默化,是基因治疗应用于人类疾病治疗的一大障碍。项目...常用的基因治疗表达载体有病毒表达载体和质粒表达载体,这两类传统基因治疗载体含有大量病毒或细菌DNA序列,会引发人体较严重的免疫反应、细胞炎症、细胞毒性副作用、以及基因表达沉默化,是基因治疗应用于人类疾病治疗的一大障碍。项目构建了一种新型的、基于腺相关病毒(AAV)倒置末端重复序列(ITR)的基因表达单链微载体(AAV-ITR mini vector),并用GFP基因作为报告基因。通过热变性的方法制备单链DNA,然后将带有GFP的质粒、双链DNA载体、AAV-ITR基因表达微载体转入真核表达细胞,采用荧光显微镜观察和流式细胞仪检测等较为简单的方法来检测其表达效率。实验结果显示,AAV-ITR基因表达微载体在293T细胞中具有较高的转染、表达效率,并且具有类似AAV病毒载体的特性。该研究结果将有助于进一步研发类似于AAV病毒载体的安全、无免疫原性的人造基因治疗载体。展开更多
Genome editing through adeno-associated viral(AAV) vectors is a promising gene therapy strategy for various diseases,especially genetic disorders. However, homologous recombination(HR) efficiency is extremely low in a...Genome editing through adeno-associated viral(AAV) vectors is a promising gene therapy strategy for various diseases,especially genetic disorders. However, homologous recombination(HR) efficiency is extremely low in adult animal models. We assumed that increasing AAV transduction efficiency could increase genome editing activity, especially HR efficiency, for in vivo gene therapy. Firstly, a mouse phenylketonuria(PKU) model carrying a pathogenic R408W mutation in phenylalanine hydroxylase(Pah) was generated. Through co-delivery of the general AAV receptor(AAVR), we found that AAVR could dramatically increase AAV transduction efficiency in vitro and in vivo. Furthermore, co-delivery of SaCas9/sgRNA/donor templates with AAVR via AAV8 vectors increased indel rate over 2-fold and HR rate over 15-fold for the correction of the single mutation in Pah;mice. Moreover, AAVR co-injection successfully increased the site-specific insertion rate of a 1.4 kb Pah cDNA by 11-fold, bringing the HR rate up to 7.3% without detectable global off-target effects. Insertion of Pah cDNA significantly decreased the Phe level and ameliorated PKU symptoms. This study demonstrates a novel strategy to dramatically increase AAV transduction which substantially enhanced in vivo genome editing efficiency in adult animal models, showing clinical potential for both conventional and genome editing-based gene therapy.展开更多
文摘Background and aim Hepatic ischemia–reperfusion injury(IRI)is a significant challenge in liver transplantation,trauma,hypovolemic shock,and hepatectomy,with limited effective interventions available.This study aimed to investigate the role of leukocyte cell-derived chemotaxin 2(LECT2)in hepatic IRI and assess the therapeutic potential of Lect2-short hairpin RNA(shRNA)delivered through adeno-associated virus(AAV)vectors.Materials and methods This study analyzed human liver and serum samples from five patients undergoing the Pringle maneuver.Lect2-knockout and C57BL/6J mice were used.Hepatic IRI was induced by clamping the hepatic pedicle.Treatments included recombinant human LECT2(rLECT2)and AAV-Lect2-shRNA.LECT2 expression levels and serum biomarkers including alanine aminotransferase(ALT),aspartate aminotransferase(AST),creatinine,and blood urea nitrogen(BUN)were measured.Histological analysis of liver necrosis and quantitative reverse-transcription polymerase chain reaction were performed.Results Serum and liver LECT2 levels were elevated during hepatic IRI.Serum LECT2 protein and mRNA levels increased post reperfusion.Lect2-knockout mice had reduced weight loss;hepatic necrosis;and serum ALT,AST,creatinine,and BUN levels.rLECT2 treatment exacerbated weight loss,hepatic necrosis,and serum biomarkers(ALT,AST,creatinine,and BUN).AAV-Lect2-shRNA treatment significantly reduced weight loss,hepatic necrosis,and serum biomarkers(ALT,AST,creatinine,and BUN),indicating therapeutic potential.Conclusions Elevated LECT2 levels during hepatic IRI increased liver damage.Genetic knockout or shRNA-mediated knockdown of Lect2 reduced liver damage,indicating its therapeutic potential.AAV-mediated Lect2-shRNA delivery mitigated hepatic IRI,offering a potential new treatment strategy to enhance clinical outcomes for patients undergoing liver-related surgeries or trauma.
文摘常用的基因治疗表达载体有病毒表达载体和质粒表达载体,这两类传统基因治疗载体含有大量病毒或细菌DNA序列,会引发人体较严重的免疫反应、细胞炎症、细胞毒性副作用、以及基因表达沉默化,是基因治疗应用于人类疾病治疗的一大障碍。项目构建了一种新型的、基于腺相关病毒(AAV)倒置末端重复序列(ITR)的基因表达单链微载体(AAV-ITR mini vector),并用GFP基因作为报告基因。通过热变性的方法制备单链DNA,然后将带有GFP的质粒、双链DNA载体、AAV-ITR基因表达微载体转入真核表达细胞,采用荧光显微镜观察和流式细胞仪检测等较为简单的方法来检测其表达效率。实验结果显示,AAV-ITR基因表达微载体在293T细胞中具有较高的转染、表达效率,并且具有类似AAV病毒载体的特性。该研究结果将有助于进一步研发类似于AAV病毒载体的安全、无免疫原性的人造基因治疗载体。
基金partially supported by grants from the National Key R&D Program of China (2019YFA0110802)the National Natural Science Foundation of China (81670470 and 81873685)+2 种基金grants from the Shanghai Municipal Commission for Science and Technology (18411953500 and 20140900201)a grant from the Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-05-E00054)the Fundamental Research Funds for the Central Universities
文摘Genome editing through adeno-associated viral(AAV) vectors is a promising gene therapy strategy for various diseases,especially genetic disorders. However, homologous recombination(HR) efficiency is extremely low in adult animal models. We assumed that increasing AAV transduction efficiency could increase genome editing activity, especially HR efficiency, for in vivo gene therapy. Firstly, a mouse phenylketonuria(PKU) model carrying a pathogenic R408W mutation in phenylalanine hydroxylase(Pah) was generated. Through co-delivery of the general AAV receptor(AAVR), we found that AAVR could dramatically increase AAV transduction efficiency in vitro and in vivo. Furthermore, co-delivery of SaCas9/sgRNA/donor templates with AAVR via AAV8 vectors increased indel rate over 2-fold and HR rate over 15-fold for the correction of the single mutation in Pah;mice. Moreover, AAVR co-injection successfully increased the site-specific insertion rate of a 1.4 kb Pah cDNA by 11-fold, bringing the HR rate up to 7.3% without detectable global off-target effects. Insertion of Pah cDNA significantly decreased the Phe level and ameliorated PKU symptoms. This study demonstrates a novel strategy to dramatically increase AAV transduction which substantially enhanced in vivo genome editing efficiency in adult animal models, showing clinical potential for both conventional and genome editing-based gene therapy.
