Intracellular delivery of biologicals such as peptides,proteins,and nucleic acids presents a great opportunity for innovative therapeutics.However,the endosome entrapment remains a major bottleneck in the intracellula...Intracellular delivery of biologicals such as peptides,proteins,and nucleic acids presents a great opportunity for innovative therapeutics.However,the endosome entrapment remains a major bottleneck in the intracellular delivery of biomacromolecules,largely limiting their therapeutic potential.Here,we converted a cell-penetrating peptide(CPP),low molecular weight protamine(LMWP),to endosomal escape peptides(EEPs)by masking LMWP with a pH-responsive counter-ionic peptide.The resulting masked CPPs(mLMWP and mLMWP2)effectively promoted the escape of peptide/protein cargoes from endosomes into the cytoplasm.Consequential lysosome repair and lysophagy were initiated upon the endolysosomal leakage.Minimal reactive oxygen species(ROS)elevation or cell death was observed.Based on mLMWP2,we constructed an intracellular protein delivery system containing an antibody as a targeting module,mLMWP2 as an endosomal escape module,and the desired protein cargo.With the HER2-targeting delivery system,we efficiently translocated cyclization recombination enzyme(Cre)and BH3-interacting domain death agonist(BID)into the cytosol of HER2^(+)cells to exert their biological activity.Thereby,the modular delivery system shows its potential as a promising tool for scientific studies and therapeutic applications.展开更多
Pluripotent stem cells(PSCs),characterized by self-renewal and capacity of differentiating into three germ layers,are the programmable building blocks of life.PSC-derived cells and multicellular systems,particularly o...Pluripotent stem cells(PSCs),characterized by self-renewal and capacity of differentiating into three germ layers,are the programmable building blocks of life.PSC-derived cells and multicellular systems,particularly organoids,exhibit great potential for regenerative medicine.However,this field is still in its infancy,partly due to limited strategies to robustly and precisely control stem cell behaviors,which are tightly regulated by inner gene regulatory networks in response to stimuli from the extracellular environment.Synthetic receptors and genetic circuits are powerful tools to customize the cellular sense-and-response process,suggesting their underlying roles in precise control of cell fate decision and function reconstruction.Herein,we review the progress and challenges needed to be overcome in the fields of PSC-based cell therapy and multicellular system generation,respectively.Furthermore,we summarize several well-established synthetic biology tools and their applications in PSC engineering.Finally,we highlight the challenges and perspectives of harnessing synthetic biology to PSC engineering for regenerative medicine.展开更多
基金supported by supported by Beijing Municipal Science&Technology Commission(Z231100007223008,China)the National Key R&D Program of China(2017YFA0207900,China)+1 种基金Tsinghua University Initiative Scientific Research Program(2023Z11DSZ001,China)the Tsinghua-Peking Joint Center for Life Sciences.
文摘Intracellular delivery of biologicals such as peptides,proteins,and nucleic acids presents a great opportunity for innovative therapeutics.However,the endosome entrapment remains a major bottleneck in the intracellular delivery of biomacromolecules,largely limiting their therapeutic potential.Here,we converted a cell-penetrating peptide(CPP),low molecular weight protamine(LMWP),to endosomal escape peptides(EEPs)by masking LMWP with a pH-responsive counter-ionic peptide.The resulting masked CPPs(mLMWP and mLMWP2)effectively promoted the escape of peptide/protein cargoes from endosomes into the cytoplasm.Consequential lysosome repair and lysophagy were initiated upon the endolysosomal leakage.Minimal reactive oxygen species(ROS)elevation or cell death was observed.Based on mLMWP2,we constructed an intracellular protein delivery system containing an antibody as a targeting module,mLMWP2 as an endosomal escape module,and the desired protein cargo.With the HER2-targeting delivery system,we efficiently translocated cyclization recombination enzyme(Cre)and BH3-interacting domain death agonist(BID)into the cytosol of HER2^(+)cells to exert their biological activity.Thereby,the modular delivery system shows its potential as a promising tool for scientific studies and therapeutic applications.
基金supported by grants from the National Key Research and Development Program(2019YFA0903800,2019YFA0110800 to W.L.,2022YFA0806302 to S.W.)the National Natural Science Foundation of China(32225030 to W.L.)the CAS Project for Young Scientists in Basic Research(YSBR-012 to W.L.).
文摘Pluripotent stem cells(PSCs),characterized by self-renewal and capacity of differentiating into three germ layers,are the programmable building blocks of life.PSC-derived cells and multicellular systems,particularly organoids,exhibit great potential for regenerative medicine.However,this field is still in its infancy,partly due to limited strategies to robustly and precisely control stem cell behaviors,which are tightly regulated by inner gene regulatory networks in response to stimuli from the extracellular environment.Synthetic receptors and genetic circuits are powerful tools to customize the cellular sense-and-response process,suggesting their underlying roles in precise control of cell fate decision and function reconstruction.Herein,we review the progress and challenges needed to be overcome in the fields of PSC-based cell therapy and multicellular system generation,respectively.Furthermore,we summarize several well-established synthetic biology tools and their applications in PSC engineering.Finally,we highlight the challenges and perspectives of harnessing synthetic biology to PSC engineering for regenerative medicine.
