Fractures continue to be a global economic burden as there are currently no osteoanabolic drugs approved to accelerate fracture healing.In this study,we aimed to develop an osteoanabolic therapy which activates the W...Fractures continue to be a global economic burden as there are currently no osteoanabolic drugs approved to accelerate fracture healing.In this study,we aimed to develop an osteoanabolic therapy which activates the Wnt/β-catenin pathway,a molecular driver of endochondral ossification.We hypothesize that using an mRNAbased therapeutic encodingβ-catenin could promote cartilage to bone transformation formation by activating the canonical Wnt signaling pathway in chondrocytes.To optimize a delivery platform built on recent advancements in liposomal technologies,two FDA-approved ionizable phospholipids,DLin-MC3-DMA(MC3)and SM-102,were used to fabricate unique ionizable lipid nanoparticle(LNP)formulations and then tested for transfection efficacy both in vitro and in a murine tibia fracture model.Using firefly luciferase mRNA as a reporter gene to track and quantify transfection,SM-102 LNPs showed enhanced transfection efficacy in vitro and prolonged transfection,minimal fracture interference and no localized inflammatory response in vivo over MC3 LNPs.The generatedβ-cateninGOF mRNA encapsulated in SM-102 LNPs(SM-102-β-cateninGOF mRNA)showed bioactivity in vitro through upregulation of downstream canonical Wnt genes,axin2 and runx2.When testing SM-102-β-cateninGOF mRNA therapeutic in a murine tibia fracture model,histomorphometric analysis showed increased bone and decreased cartilage composition with the 45μg concentration at 2 weeks post-fracture.μCT testing confirmed that SM-102-β-cateninGOF mRNA promoted bone formation in vivo,revealing significantly more bone volume over total volume in the 45μg group.Thus,we generated a novel mRNA-based therapeutic encoding aβ-catenin mRNA and optimized an SM-102-based LNP to maximize transfection efficacy with a localized delivery.展开更多
Blocking the programmed death-ligand 1(PD-L1)on tumor cells with monoclonal antibody therapy has emerged as powerful weapon in cancer immunotherapy.However,only a minority of patients presented immune responses in cli...Blocking the programmed death-ligand 1(PD-L1)on tumor cells with monoclonal antibody therapy has emerged as powerful weapon in cancer immunotherapy.However,only a minority of patients presented immune responses in clinical trials.To develop an alternative treatment method based on immune checkpoint blockade,we designed a novel and efficient CRISPR-Cas9 genome editing system delivered by cationic copolymer aPBAE to downregulate PD-L1 expression on tumor cells via specifically knocking out Cyclin-dependent kinase 5(Cdk5)gene in vivo.The expression of PD-L1 on tumor cells was significantly attenuated by knocking out Cdk5,leading to effective tumor growth inhibition in murine melanoma and lung metastasis suppression in triple-negative breast cancer.Importantly,we demonstrated that aPBAE/Cas9-Cdk5 treatment elicited strong T cell-mediated immune responses in tumor microenvironment that the population of CD8^+T cells was significantly increased while regulatory T cells(Tregs)was decreased.It may be the first case to exhibit direct in vivo PD-L1 downregulation via CRISPR-Cas9 genome editing technology for cancer therapy.It will provide promising strategy for preclinical antitumor treatment through the combination of nanotechnology and genome engineering.展开更多
基金supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases(NIAMS)of the National Institutes of Health(NIH)under award number R01 AR077761support from the Musculoskeletal Regeneration Partnership Fund by Mary Sue and Michael Shannon and by Project Number 20-166 from the Orthoregeneration Network for Kick-Starter Grantsupported by the National Institute on Aging of the National Institutes of Health under Award Number F30AG077748 and the University of Wisconsin-Madison Medical Scientist Training Program:T32GM140935.All content is solely the responsibility of the authors and does not represent the official views of National Institutes of Health,Orthoregeneration Network or Shannon Foundation.
文摘Fractures continue to be a global economic burden as there are currently no osteoanabolic drugs approved to accelerate fracture healing.In this study,we aimed to develop an osteoanabolic therapy which activates the Wnt/β-catenin pathway,a molecular driver of endochondral ossification.We hypothesize that using an mRNAbased therapeutic encodingβ-catenin could promote cartilage to bone transformation formation by activating the canonical Wnt signaling pathway in chondrocytes.To optimize a delivery platform built on recent advancements in liposomal technologies,two FDA-approved ionizable phospholipids,DLin-MC3-DMA(MC3)and SM-102,were used to fabricate unique ionizable lipid nanoparticle(LNP)formulations and then tested for transfection efficacy both in vitro and in a murine tibia fracture model.Using firefly luciferase mRNA as a reporter gene to track and quantify transfection,SM-102 LNPs showed enhanced transfection efficacy in vitro and prolonged transfection,minimal fracture interference and no localized inflammatory response in vivo over MC3 LNPs.The generatedβ-cateninGOF mRNA encapsulated in SM-102 LNPs(SM-102-β-cateninGOF mRNA)showed bioactivity in vitro through upregulation of downstream canonical Wnt genes,axin2 and runx2.When testing SM-102-β-cateninGOF mRNA therapeutic in a murine tibia fracture model,histomorphometric analysis showed increased bone and decreased cartilage composition with the 45μg concentration at 2 weeks post-fracture.μCT testing confirmed that SM-102-β-cateninGOF mRNA promoted bone formation in vivo,revealing significantly more bone volume over total volume in the 45μg group.Thus,we generated a novel mRNA-based therapeutic encoding aβ-catenin mRNA and optimized an SM-102-based LNP to maximize transfection efficacy with a localized delivery.
基金supported by the National Natural Science Foundation of China(81872810,81673374 and 81871473)Wuhan University of Science and Technology Plan for Applied Fundamental Research(2017060201010146,China)the Fundamental Research Funds for the Central Universities(2018KFYYXJJ019,2019KFYRCPY049 and 2016YXMS138,China).
文摘Blocking the programmed death-ligand 1(PD-L1)on tumor cells with monoclonal antibody therapy has emerged as powerful weapon in cancer immunotherapy.However,only a minority of patients presented immune responses in clinical trials.To develop an alternative treatment method based on immune checkpoint blockade,we designed a novel and efficient CRISPR-Cas9 genome editing system delivered by cationic copolymer aPBAE to downregulate PD-L1 expression on tumor cells via specifically knocking out Cyclin-dependent kinase 5(Cdk5)gene in vivo.The expression of PD-L1 on tumor cells was significantly attenuated by knocking out Cdk5,leading to effective tumor growth inhibition in murine melanoma and lung metastasis suppression in triple-negative breast cancer.Importantly,we demonstrated that aPBAE/Cas9-Cdk5 treatment elicited strong T cell-mediated immune responses in tumor microenvironment that the population of CD8^+T cells was significantly increased while regulatory T cells(Tregs)was decreased.It may be the first case to exhibit direct in vivo PD-L1 downregulation via CRISPR-Cas9 genome editing technology for cancer therapy.It will provide promising strategy for preclinical antitumor treatment through the combination of nanotechnology and genome engineering.
