AIM To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout(Fah-/-)mice by homologous-recombination-mediated targeted addition of the Fah gene.METHODS C57 BL/6 Fah?exon5 mice s...AIM To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout(Fah-/-)mice by homologous-recombination-mediated targeted addition of the Fah gene.METHODS C57 BL/6 Fah?exon5 mice served as an animal model for human tyrosinaemia type 1 in our study.The vector was created by amplifying human Fah c DNA including the TTR promoter from a lentivirus plasmid as described.The Fah expression cassette was flanked by homologous arms(620 bp and 749 bp long)of the Rosa26 gene locus.Mice were injected with 2.1×108 VP of this vector(r AAV8-ROSA26.HAL-TTR.FahROSA26.HAR)via the tail vein.Mice in the control group were injected with 2.1×108 VP of a similar vector but missing the homologous arms(r AAV8-TTR.Fah).Primary hepatocytes from Fah-/-recipient mice,treated with our vectors,were isolated and 1×106 hepatocytes were transplanted into secondary Fah-/-recipient mice by injection into the spleen.Upon either vector application or hepatocyte transplantation NTBC treatment was stopped in recipient mice.RESULTS Here,we report successful HR-mediated genome editing by integration of a Fah gene expression cassette into the"safe harbour locus"Rosa26 by recombinant AAV8.Both groups of mice showed long-term survival,weight gain and FAH positive clusters as determined by immunohistochemistry analysis of liver sections in the absence of NTBC treatment.In the group of C57 BL/6 Fah?exon5 mice,which have been transplanted with hepatocytes from a mouse injected with r AAV8-ROSA26.HAL-TTR.Fah-ROSA26.HAR 156 d before,6 out of 6 mice showed long-term survival,weight gain and FAH positive clusters without need for NTBC treatment.In contrast only 1 out 5 mice,who received hepatocytes from r AAV8-TTR.Fah treated mice,survived and showed few and smaller FAH positive clusters.These results demonstrate that homologous recombinationmediated Fah gene transfer corrects the phenotype in a mouse model of human tyrosinaemia type 1(Fah-/-mice)and is long lasting in a proliferating state of the liver as shown by withdrawal of NTBC treatment and serial transplantation of isolated hepatocytes from primary Fah-/-recipient mice into secondary Fah-/-recipient mice.This long term therapeutic efficacy is clearly superior to our control mice treated with episomal r AAV8 gene therapy approach.CONCLUSION HR-mediated r AAV8 gene therapy provides targeted transgene integration and phenotypic correction in Fah-/-mice with superior long-term efficacy compared to episomal r AAV8 therapy in proliferating livers.展开更多
A recent study published in Nature by Maya M.Arce and colleagues unveils the role of central gene circuits in governing the delicate balance between T cell rest and T cell activation.This work bridges fundamental mole...A recent study published in Nature by Maya M.Arce and colleagues unveils the role of central gene circuits in governing the delicate balance between T cell rest and T cell activation.This work bridges fundamental molecular biology with findings in preclinical models,providing insights into context-specific gene regulation and potential therapeutic targets for immune modulation.展开更多
Human induced pluripotent stem cell-derived neural stem/progenitor cells are used in cell-replacement and regenerative therapeutic strategies after traumatic central nervous system injury.Traumatic injury alters the h...Human induced pluripotent stem cell-derived neural stem/progenitor cells are used in cell-replacement and regenerative therapeutic strategies after traumatic central nervous system injury.Traumatic injury alters the host microenvironment,which in turn affects the functionality of transplanted human neural stem/progenitor cells and potentially limits their benefits for neurorepair.However,the underlying mechanisms through which the host environment alters the fate and functionality of transplanted human neural stem/progenitor cells remain poorly understood.Here,we showed that massive deposition of blood-derived fibrinogen in a mouse model of spinal cord injury contributed to an altered lesion environment.Fibrinogen promoted human neural stem/progenitor cell differentiation into reactive astrocytes by activating the BMP receptor signaling pathway and inducing of the transcriptional regulator inhibitor of DNA binding 3.ID3-depleted human neural stem/progenitor cells,generated by CRISPR/Cas9-mediated genome editing,reduced astrocyte formation in response to astrogenic stimuli.Instead,ID3-depleted human neural stem/progenitor cells had a bipolar,immature glial progenitor cell phenotype.These modified cells secreted extracellular vesicles with a distinct miRNA profile that enhanced neurite outgrowth.We conclude that targeting inhibitor of DNA binding 3 in human neural stem/progenitor cells can beneficially modulate their functionality and cell fate in the injured central nervous system toward glial progenitor cells,potentially enhancing their capacity to promote central nervous system repair.展开更多
基金SFB 738the"Deutsche Forschungsgemeinschaft"(Gerok-Grant)for financial support
文摘AIM To stably correct tyrosinaemia in proliferating livers of fumarylacetoacetate-hydrolase knockout(Fah-/-)mice by homologous-recombination-mediated targeted addition of the Fah gene.METHODS C57 BL/6 Fah?exon5 mice served as an animal model for human tyrosinaemia type 1 in our study.The vector was created by amplifying human Fah c DNA including the TTR promoter from a lentivirus plasmid as described.The Fah expression cassette was flanked by homologous arms(620 bp and 749 bp long)of the Rosa26 gene locus.Mice were injected with 2.1×108 VP of this vector(r AAV8-ROSA26.HAL-TTR.FahROSA26.HAR)via the tail vein.Mice in the control group were injected with 2.1×108 VP of a similar vector but missing the homologous arms(r AAV8-TTR.Fah).Primary hepatocytes from Fah-/-recipient mice,treated with our vectors,were isolated and 1×106 hepatocytes were transplanted into secondary Fah-/-recipient mice by injection into the spleen.Upon either vector application or hepatocyte transplantation NTBC treatment was stopped in recipient mice.RESULTS Here,we report successful HR-mediated genome editing by integration of a Fah gene expression cassette into the"safe harbour locus"Rosa26 by recombinant AAV8.Both groups of mice showed long-term survival,weight gain and FAH positive clusters as determined by immunohistochemistry analysis of liver sections in the absence of NTBC treatment.In the group of C57 BL/6 Fah?exon5 mice,which have been transplanted with hepatocytes from a mouse injected with r AAV8-ROSA26.HAL-TTR.Fah-ROSA26.HAR 156 d before,6 out of 6 mice showed long-term survival,weight gain and FAH positive clusters without need for NTBC treatment.In contrast only 1 out 5 mice,who received hepatocytes from r AAV8-TTR.Fah treated mice,survived and showed few and smaller FAH positive clusters.These results demonstrate that homologous recombinationmediated Fah gene transfer corrects the phenotype in a mouse model of human tyrosinaemia type 1(Fah-/-mice)and is long lasting in a proliferating state of the liver as shown by withdrawal of NTBC treatment and serial transplantation of isolated hepatocytes from primary Fah-/-recipient mice into secondary Fah-/-recipient mice.This long term therapeutic efficacy is clearly superior to our control mice treated with episomal r AAV8 gene therapy approach.CONCLUSION HR-mediated r AAV8 gene therapy provides targeted transgene integration and phenotypic correction in Fah-/-mice with superior long-term efficacy compared to episomal r AAV8 therapy in proliferating livers.
文摘A recent study published in Nature by Maya M.Arce and colleagues unveils the role of central gene circuits in governing the delicate balance between T cell rest and T cell activation.This work bridges fundamental molecular biology with findings in preclinical models,providing insights into context-specific gene regulation and potential therapeutic targets for immune modulation.
基金supported by a Fill in the Gap fellowship(Medical Faculty Freiburg)(to JDL)a Fritz Thyssen Stiftung grant,a European Stroke Research Foundation(ESRF)grant,a Wings for Life foundation grantthe DFG grants SCHA 1442/8-1,SCHA 1442/8-3,and SCHA 1442/9-1(to CS).
文摘Human induced pluripotent stem cell-derived neural stem/progenitor cells are used in cell-replacement and regenerative therapeutic strategies after traumatic central nervous system injury.Traumatic injury alters the host microenvironment,which in turn affects the functionality of transplanted human neural stem/progenitor cells and potentially limits their benefits for neurorepair.However,the underlying mechanisms through which the host environment alters the fate and functionality of transplanted human neural stem/progenitor cells remain poorly understood.Here,we showed that massive deposition of blood-derived fibrinogen in a mouse model of spinal cord injury contributed to an altered lesion environment.Fibrinogen promoted human neural stem/progenitor cell differentiation into reactive astrocytes by activating the BMP receptor signaling pathway and inducing of the transcriptional regulator inhibitor of DNA binding 3.ID3-depleted human neural stem/progenitor cells,generated by CRISPR/Cas9-mediated genome editing,reduced astrocyte formation in response to astrogenic stimuli.Instead,ID3-depleted human neural stem/progenitor cells had a bipolar,immature glial progenitor cell phenotype.These modified cells secreted extracellular vesicles with a distinct miRNA profile that enhanced neurite outgrowth.We conclude that targeting inhibitor of DNA binding 3 in human neural stem/progenitor cells can beneficially modulate their functionality and cell fate in the injured central nervous system toward glial progenitor cells,potentially enhancing their capacity to promote central nervous system repair.
