Design And Construction Of Precisely-controlled Protein Secretion System And Its Application Research

The development of controllable gene circuits based on synthetic biology principles has provided a safe and efficient genetic control system for intelligent gene therapy and cell therapy.Engineered cells equipped with theses inducible gene circuits can sense user-defined external regulatory signals and precisely regulate the expression and secretion of therapeutic proteins in a predictable and intelligent manner,driving disease treatment towards a safer,personalized,precise,and efficient direction.However,currently available controllable gene circuits typically rely on the regulation of transcription or translation,which often results in a time delay of hours or even days before a sufficient amount of therapeutic proteins is released in response to induced signals.The slow response speed poses two main issues:1)it doesn’t align with the rapid release rate of normal cells in response to external inducers.Under physiological conditions,many cells respond very quickly to external inducers,such as pancreatic cells releasing insulin in response to elevated blood sugar levels.2)it is not suitable for many diseases that require rapid treatment,such as asthma,acute myocardial infarction,angina pectoris,etc.Therefore,this study focuses on designing and constructing a precise and controllable rapid protein secretion system based on synthetic biology principles,which can respond promptly to external signals and release therapeutic proteins within minutes,providing a highly sensitive and precisely controllable platform for the rapid and instantaneous delivery of protein drugs in intelligent gene therapy and cell therapy.In the classical protein secretion process,there is a subset of proteins that,after translation,do not undergo release into the extracellular space.Instead,they are retained within the endoplasmic reticulum due to the presence of a retrieval signal at their terminus.Leveraging this protein sorting principle,we devised and constructed a protease-based rapid protein secretion system(PASS).We incorporated an endoplasmic reticulum retrieval signal at the end of the target protein and introduced a peptide chain recognized by a protease between the retrieval signal and the target protein.By utilizing a protease to cleave the retrieval signal,we successfully achieved controlled target protein secretion.To achieve precise and controlled regulation of target protein secretion,we developed three chemical-inducible protein secretion system(ChemPASS).These systems utilize a split tobacco etch virus protease,split-TEVp,whose activity is regulated through small-molecule-mediated heterodimerization.ChemPASS systems offer a high degree of adjustability and efficiency.They can selectively respond to citrate,as well as two clinically approved drugs,inducing rapid secretion of the target protein within minutes.In line with the principles of tumor immunotherapy,we next engineered a tumorantigen-triggered protein secretion system(AntigenPASS)to sense antigens and subsequently trigger the local secretion of cytokines which induced cell apoptosis.We here took advantage of synNotch receptor,anchored the TEVp protease to the cell membrane.Target antigen binding leads to release the intracellular TEVp domain from its tether,bringing TEVp into proximity of TEVcs,thus triggering protein secretion.AntigenPASS can identify different antigens and,by replacing the reporter protein,achieve controlled release of various cytotoxic proteins and immune regulators.It enables precise tumor cell targeting,introducing a novel approach to immunotherapy.Leveraging the advantages of optogenetics,we developed an optogenetic controlled rapid protein secretion system(OptoPASS).We achieved remote,traceless,highly spatial and temporal specific regulation of protein secretion by utilizing Magnets-based blue light-mediated dimerization system to control the activity of the split-TVMVp protease from the Tobacco Vein Mottling Virus.It can induce protein secretion effectively within just 5 minutes of light exposure.Furthermore,w we have demonstrated that OptoPASS can secrete therapeutic proteins within minutes in various disease model mice.In diabetic,hypertensive,and pain murine model,as little as 15 minutes of light exposure led to reductions in blood glucose,blood pressure,and alleviation of pain,underscoring its potential for clinical application.Collectively,we developed protease-based rapid protein secretion system that can be customized to drive user-defined therapeutic protein secretion within minutes in response to external signal inputs.The PASS system showcases adaptability and flexibility,coupled with the capability for swift responses to external stimuli and the quick release of therapeutic proteins within minutes.PASS offers an innovative genetic control system for precise and controllable gene-based and cell-based therapy.